15
J. Anat. (1967), 101, 4, pp. 833-S47 833 Printed in Great Britain Proceedings of the Anatomical Society of Great Britain and Ireland FEBRUARY 1967 An Ordinary Meeting of the Society for the Session 1966-67 was held on Friday 24 February 1967 at the Department of Anatomy, Middlesex Hospital Medical School, London. The President (Professor D. V. Davies) and Professors P. H. S. Silver and G. M. Wyburn occupied the Chair at the various sessions. The following are the authors' abstracts of papers presented. Lens formation from the optic vesicle in chick and duck embryos. By P. H. S. SILVER, Middlesex Hospital Medical School Colucci (Memorie R. Accad. Sci. Ist. Bologna, series 5, 1, 1891) showed that removal of the lens from newt eyes was followed by regeneration of a new lens from the dorsal border of the iris (Wolffian regeneration). There is at present no agreement as to the direct lens-forming capacity of the optic vesicle or optic cup in bird embryos. The present communication presents new evidence indicating that the optic vesicle has lens-generating capacity. Primary optic vesicles were denuded of their overlying presumptive lens epithelium and adjacent ectoderm by trypsinization. Histological examination of forty-five embryos showed that no islands of head-surface epithelium remained attached to the vesicle. The graft vesicles were transferred isotopically (fifty-two experiments) to a host of different species (chick in duck or duck in chick). Hosts continued development in ovo, and the lenses were harvested at about stage 30. Nineteen experiments succeeded in producing a well-formed eye (eight chick in duck, eleven duck in chick). In the remaining thirty-three the eyes were very small or grossly abnormal, the lenses absent or too small to identify for certain in the gross specimens. The species of origin (duck or chick) of the nineteen test lenses was demonstrated by the Ouchterlony technique to determine whether they had originated from the graft or from the host. They were all shown to be of donor origin. These test lens could have developed in one of three ways: either (1) from an island of pre- sumptive lens cells carried over from the donor; or (2) from neo-induction of host epithelium by the graft vesicle; or (3) from the graft vesicle itself, by a process of Wolffian style generation. The evidence favours the last possibility. Observations on the fine structure of the outer ectodermal cells of the gastrulae of Xenopus laevis. By F. BILLET and R. P. GOULD, University of Southampton and the Middlesex Hospital Medical School. (Figs. 1-2) The first cell type to be clearly differentiated during the development of the Xenopus embryo is found in the outermost layer of cells. The cytological differentiation consists of a polarization of the cell organelles, and the development of an apical border complex that consists of large vesicles (Fig. 1). These vesicles are arranged just under the apical plasma membrane and many of them contain a densely basophilic material. The fine structure of these early epidermal cells indicates that the vesicles are derived from granulated cisternae of the endoplasmic reticulum (Fig. 2). A flocculent material of increasing electron density accumulates in the cistenae which gradually enlarge (Fig. 2) to form vesicles. 52-2

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Page 1: Proceedings of the Anatomical Society of Great Britain and Ireland

J. Anat. (1967), 101, 4, pp. 833-S47 833Printed in Great Britain

Proceedings of the Anatomical Society ofGreat Britain and Ireland

FEBRUARY 1967

An Ordinary Meeting of the Society for the Session 1966-67 was held on Friday 24 February1967 at the Department of Anatomy, Middlesex Hospital Medical School, London.The President (Professor D. V. Davies) and Professors P. H. S. Silver and G. M. Wyburn

occupied the Chair at the various sessions.The following are the authors' abstracts of papers presented.

Lens formation from the optic vesicle in chick and duck embryos. By P. H. S. SILVER,Middlesex Hospital Medical School

Colucci (Memorie R. Accad. Sci. Ist. Bologna, series 5, 1, 1891) showed that removal of thelens from newt eyes was followed by regeneration of a new lens from the dorsal border of theiris (Wolffian regeneration).

There is at present no agreement as to the direct lens-forming capacity of the optic vesicleor optic cup in bird embryos. The present communication presents new evidence indicatingthat the optic vesicle has lens-generating capacity.

Primary optic vesicles were denuded of their overlying presumptive lens epithelium andadjacent ectoderm by trypsinization. Histological examination of forty-five embryos showedthat no islands of head-surface epithelium remained attached to the vesicle. The graft vesicleswere transferred isotopically (fifty-two experiments) to a host of different species (chick in duckor duck in chick). Hosts continued development in ovo, and the lenses were harvested at aboutstage 30.

Nineteen experiments succeeded in producing a well-formed eye (eight chick in duck, elevenduck in chick). In the remaining thirty-three the eyes were very small or grossly abnormal, thelenses absent or too small to identify for certain in the gross specimens.The species of origin (duck or chick) of the nineteen test lenses was demonstrated by the

Ouchterlony technique to determine whether they had originated from the graft or from thehost. They were all shown to be of donor origin.These test lens could have developed in one of three ways: either (1) from an island of pre-

sumptive lens cells carried over from the donor; or (2) from neo-induction of host epitheliumby the graft vesicle; or (3) from the graft vesicle itself, by a process of Wolffian style generation.The evidence favours the last possibility.

Observations on the fine structure of the outer ectodermal cells of the gastrulae ofXenopus laevis. By F. BILLET and R. P. GOULD, University of Southampton and theMiddlesex Hospital Medical School. (Figs. 1-2)

The first cell type to be clearly differentiated during the development of the Xenopus embryois found in the outermost layer of cells. The cytological differentiation consists of a polarizationof the cell organelles, and the development of an apical border complex that consists of largevesicles (Fig. 1). These vesicles are arranged just under the apical plasma membrane and manyof them contain a densely basophilic material.The fine structure of these early epidermal cells indicates that the vesicles are derived from

granulated cisternae of the endoplasmic reticulum (Fig. 2). A flocculent material of increasingelectron density accumulates in the cistenae which gradually enlarge (Fig. 2) to form vesicles.

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Proceedings of the Anatomical Society ofThe ribosomes appear to become detached from the larger vesicles which then migrate to thefree border of the cell. Their flocculent material becomes condensed and may be dischargedinto the surrounding medium.

The action of tissue homogenates on the early chick embryo and on chick fibroblastsin culture. By J. McKENZIE and H. ALLAM, University of Aberdeen

Earlier communications have described the effects of tissue homogenates upon the explantedchick embryo of 24-28 h incubation: the main features were a general stunting of growth, thepresence of a dense ring of tissue at the periphery of the disc and necrosis of the neural tube.Treatment of the homogenate with DNase did not alter its effect on the embryo whereas heatingto 60 'C or treatment with RNase made the homogenate more effective. Different methods forthe extraction of RNA have now been applied to the tissue homogenates and the results suggestthat RNA or some part of the RNA molecule is the active ingredient of the tissues. Digestionof the extract with RNase increases its effect on the embryo although heating it causes nosignificant change. Preliminary quantitative estimations of the amount of RNA required foreach embryo have been made and the most susceptible stages of early chick embryonic develop-ment have been determined.The same extract when added to chick fibroblasts in culture has produced well-defined and

consistent morphological changes which persist after the cells are returned to untreated culturemedium. These alterations in the state of differentiation of cells in culture may help to throwlight on the changes seen in the early chick embryo where there are cells at varying stages ofdifferentiation.

The effect of saline loading on the development of the prefunctional salt gland in the domesticduck. By T. LUNN and A. D. HALLY, University of Newcastle upon Tyne

The salt glands of the domestic duck, like those of marine birds, are osmoregulatory organswhich secrete a hypertonic saline in response to an osmotic load (Scothorne, Q. Jl exp. Physiol.44, 1959). Secretory capacitance first develops 3 days before hatching, after about 241 d ofincubation (Hally, Buxton & Scothorne, J. Anat. 100, 1966).The functional demand of a prolonged osmotic load causes growth in the adult salt gland

(Benson & Phillips, J. Anat. 98, 1964), and this investigation is an attempt to determine ifa functional demand affects the development of the prefunctional gland.

Aylesbury duck eggs were loaded osmotically on either 14th or 20th d of incubation byinjecting 10 Y NaCl solution (20 m-equiv NaCl/kg egg) into the albumen sac. The effectivenessof the load was assessed by subsequent plasma chloride estimations. Incubation was continueduntil day 244, when the secretary capacities of these salt glands were measured and comparedwith those from control embryos. The secretary capacity was determined by estimating thechloride content of the secretion provoked by an intravenous salt load (20 M-equiv NaCl/kg egg).The mean secretary output, in ,t-equiv Cl/kg embryo/min, was 1-41± S.E. 0-41 -for the 14 dexperimental group, and 1-48 ± S.E. 0-26 for the 20 d experimental group. Both these means aresignificantly greater than that of the controls, which was 0 65 ± S.E. 0 12.

These results indicate that a prolonged functional demand accelerates the development of theprefunctional salt gland.

The relationship between nucleolar size and RNA synthesis in cells of thehair cortex of rats. By R. T. SIMs, University of Cambridge

It has not been possible to demonstrate nucleoli in cortical cells of the upper keratogenouszone in rat hair follicles with specific stains for RNA. Because the nucleolus is thought to beconcerned with RNA synthesis an experiment was performed on the incorporation of 3H-cytidineinto the anucleolate cells of the hair cortex.Twelve albino rats were injected subcutaneously with 0 4 mc/100 g body weight 3H-cytidine

(specific activity 990 mc/mM). They were killed in pairs 1, 3, 12, 24, 48 and 72 h after the

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Great Britain and Ireland 835

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Proceedings of the Anatomical Society ofinjection. Specimens of skin bearing actively growing hair were taken and fixed in Carnoy'sfluid. Autoradiographs were prepared with Kodak AR 10 stripping film. Good autoradiographswere obtained after 41 d exposure of the sections to the photographic emulsion.

In the specimens taken 1 h and 3 h after the injection only one group of nuclei are notdemonstrably radioactive. They are the cortical cells above the mid-keratogenous zone of thefollicles. The density of silver grains in the photographic emulsion over nuclei of the bulb ishigh. This shows that RNA synthesis is greatly reduced in the anucleolate cells.The cytoplasm of the cortical cells up to the end of the keratogenous zone is radioactive in the

12, 24 and 72 h specimens. Because the cells continuously move along the follicles from thematrix, RNA synthesized in the matrix is carried to higher levels. We know that cell divisionoccurs only in the matrix, increase in cell size occurs only in the upper bulb and differentproteins are manufactured at the top and bottom of the keratogenous zone. It must be concludedthat some RNA is associated with each of these different activities in turn and one can speculatethat it is non-specific ribosomal RNA.

Enzyme histochemistry of the pars intermedia of the ox pituitary.By A. T. RAFTERY, University ofLeeds

Little is known about the pars intermedia of the pituitary gland in mammals, and althoughnumerous histochemical studies of the other divisions of the gland have been published thereare few specifically concerned with this part. This study deals with the localization of severalenzymes in the hope that such knowledge might help in elucidating the functions of the parsintermedia. The enzymes studied were: alkaline phosphatase, acid phosphatase, adenosinetriphosphatase, non-specific esterase, cholinesterases, succinic dehydrogenase and cytochromeoxidase. Appropriate inhibitors and control sections were used.

Alkaline phosphatase and adenosine triphosphatase were present in the walls of the largervessels of the interlobular connective tissue. Adenosine triphosphatase alone was present inthe walls of the capillaries of the pars intermedia. The epithelial cells of the pars intermediashowed a strong positive reaction for succinic dehydrogenase and cytochrome oxidase, buta negative reaction for acid phosphatase. A non-specific esterase, identified as an A-esterase,was also present; the reaction for this enzyme was strongly positive in some cells, but weaklyso in others. A strong positive reaction for cholinesterases was present in some cells, but againothers showed a weak reaction.The presence of these various enzymes in the pars intermedia suggests that it is actively

functioning, presumably as a secretary organ. The only hormone known to be produced in thepars intermedia is melanocyte-stimulating hormone (MSH). In some cold-blooded vertebratesthis hormone controls a camouflaging mechanism, but its function in mammals is obscure.It seems probable however, that in these forms the hormone may exert actions other thana purely melanocyte-stimulating one. The close structural relationship between MSH andcorticotrophin (ACTH) and the fact that MSH, like ACTH, possesses the ability to releasefree fatty acids from depot fat suggests that MSH may have some corticotropic activity.

Esterase activity in the epidermis of rat and mouse tongue. By A. K. CANE and R. I. C.SPEARMAN, Miles Laboratories Ltd., Stoke Poges and University College HospitalMedical School

The epidermis of the rat and mouse tongue dorsal surface forms a repeating pattern offiliform papillae and trough zones between them. The papillae are composed of three partswhich can be distinguished by their histological structure and histochemical reactions. There isa central spine of hard keratinized cells, an anterior convex horny part formed over a granularlayer and a posterior part of scale-like keratin which is not formed over a granular layer. Thetrough zone between the papillae has larger and more irregular granules in the granular layerand lies in a horizontal plane as opposed to the inclined plane of the papillary granular layer.Peracetic acid oxidation followed by thioflavine T staining and examination by u.v. fluorescencehas shown that all the horny layers are rich in disulphide bonds characteristic of keratinized

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structures. Neutral red selectively stains the convex horny layer. Acid phosphatase is concen-trated in the keratogenous zone of the spine in the mouse and rat but hardly any is found inthe transitional zone of the trough and granular layer regions. The rat basal layer containsacid phosphatase and in the posterior part of the tongue there is a unilateral distribution in thebasal layer which may be related to the histogenesis of the papilla. Acid phosphatase is notalways associated with keratinization in the various types of tongue horny layer. The distri-bution therefore differs from that in human integumental skin. The ventral surface of boththese animals' tongues is parakeratotic but very little acid phosphatase is present in this hornylayer, unlike the parakeratotic-like horny layer of the mouse or rat tail scale which containsvery large amounts. The periphery of the germinative cells of the spine is rich in alkalinephosphatase. Non-specific esterase is found in the convex and concave papillary horny layersbut not in the spine or trough regions. The distribution of enzymes is probably species specific.

Lyo- and desmo-esterases in the rat placenta. By A. W. F. FISHER andD. BULMER, University of Manchester

As a preliminary to parallel investigations of rat placental esterases by cytochemical andelectrophoretic methods a study was made of the relative proportions of lyo- and desmo-enzymesin this situation.

Six fresh placentae were cut at 10 #m on the freezing microtome and the sections left in1 ml distilled water at 4 0C for 1 h. After centrifugation at 20,000 g the supernatant fluidcontained the lyo-esterase. The centrifugate was washed three times with distilled water andfrozen and thawed five times before centrifuging again at 20,000g; the final supernatant con-tained the desmo-esterase, and this was confirmed by the lack of demonstrable activity in thefinal centrifugate.

Samples of the two esterase fractions, with and without concentration, were submitted tostarch-gel electrophoresis and the strips exposed to simultaneous coupling methods with a varietyof esterase substrates. The results show that the lyo-esterase fraction of the placenta constitutesa much greater proportion of the total esterase content than the desmo-fraction. Differentsubstrates show differing zymogram patterns, but for each substrate the desmo-fraction showsthe same bands of activity as the lyo-fraction, though with much less intensity.The relatively high proportion of lyo-esterase explains the experience of cytochemical studies,

where fresh cryostat sections show only a small proportion of the esterase activity which isdemonstrated in frozen sections of formalin-fixed tissue. Fresh cryostat sections exposed towater for a few minutes before incubation in substrate mixture show still less activity. Esterasereactions on fresh cryostat sections therefore give a very inadequate picture of total esterasecontent. Differing reactions with different substrates under these conditions may be partlycorrelated with substrate differences in the electrophoretograms.

Correlation of cytochemical and electrophoretic studies on the non-specific esterasesof the rat placenta. By D. BULMER and A. W. F. FISHER, University of Manchester

Cytochemical studies of rat placental esterases, on frozen sections of formalin-fixed tissueblocks, show differing distributions of activity with different substrates. The enzyme of thedecidua basalis, which is revealed strongly with naphthyl acetate and naphthyl propionate,produces little or no demonstrable hydrolysis of naphthyl AS acetate. The metrial gland cellesterase reacts strongly with naphthol AS acetate, but shows relatively weak activity withnaphthyl acetate. Naphthol AS chloropropionate is hydrolysed weakly by the metrial glandcell granules, but there is marked staining in the endothelium of maternal vessels and in thejunctional zone trophoblast which does not occur with naphthol AS acetate.

Homogenates of rat placenta, after repeated freezing and thawing to extract both lyo- anddesmo-esterases, were submitted to starch-gel electrophoresis at pH 7-5; strips were exposedto the same histochemical methods as those employed on the tissue sections. The wide range ofbands of activity demonstrated with naphthyl acetate and naphthyl propionate contrasts with

Great Britain and Ireland 837

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Proceedings of the Anatomical Society ofthe more restricted, and mutually differing, zymogram patterns obtained with naphtholAS acetateand naphthol AS chloropropionate. The cathodally-migrating enzymes shown with naphthylAS acetate are particularly resistant to inhibition with E 600, while the anodally migratingenzymes shown with naphthol AS chloropropionate are particularly resistant to Mipafox.

The autonomic innervation of epididymal adipose tissue in the rat.By BRYAN BALLANTYNE and A. T. RAFTERY, University of Leeds

There are differences of opinion in the literature about the distribution and function of nervesin adipose tissue, and whether the autonomic supply is sympathetic, parasympathetic, or mixed.We have investigated the neurohistology of epididymal adipose tissue by cholinesterase histo-chemistry, quantitatively estimated the cholinesterase activity of the tissue, and performedbio-assays for acetylcholine (ACh).

Six adult Sprague-Dawley rats were killed by decapitation, the epididymal fat removed anddivided into three. One weighed portion was homogenized in distilled water for the estimationof acetylcholinesterase, butyrylcholinesterase, and propionylcholinesterase activities by continu-ous potentiometric titration at pH 7-4 at 37 'C; the second portion was fixed for histochemicallocalization of the cholinesterases; while the remaining portion was weighed, homogenized in104 M eserinized Ringer's solution, and used in a bio-assay for ACh (Anand, Am. J. Physiol. 168,1952). Specimens of brain and parotid salivary gland served as controls; the maximum sensitivityof the method was 103 #ug ACh/ml.The mean concentration of acetylcholinesterase was 62-7 ± 13-7 S.D. /tM/g/h, and the enzyme

was histochemically detectable only in the cytoplasma of fat cells. The mean concentration ofbutyrylcholinesterase was 68-0 ± 22-5 S.D. /M/g/h and of propionylcholinesterase was 126-1 ±22-0 S.D./,M/g/h; these enzymes were present mainly in fat cell cytoplasm, but some activitywas present in nerves arranged as peri-arterial plexuses. Nerves could not be demonstrated atany other sites, and ganglion cells were not seen.The failure to detect any ACh indicated that no cholinergic nerves are present in the tissue;

the control brain had a mean level of 2-21 ± 0-56 S.D. #ugACh/g, and the parotid salivary glanda mean of 0-62 ± 0-27 S.D.,ug AC/g in keeping with its profuse para-sympathetic innervation.

These observations suggest that epididymal adipose tissue is supplied only by post-ganglionicsympathetic fibres which are arranged as perivascular plexuses, and that the autonomic nervoussystem can directly regulate adipose tissue metabolism only by varying the flow of blood throughthe tissue.

A hominid toe bone from Bed I, Olduvai Gorge, Tanzania. By MICHAEL H. DAY,Middlesex Hospital Medical School

During the excavation of the site FLK North (FLK NI) at Olduvai Gorge a hominid toebone was recovered on 16 November 1961. The bone was found at Level 5, near the top ofBed 1, and it was associated with implements of the Oldowan culture as well as numerousbroken mammalian bones. No other hominid remains were found at this site, which is regardedas being of Upper Villafranchian age.The bone is the right hallucial terminal phalanx of an adult hominid; it is complete and

undistorted. Previous morphological and functional assessment of the bone has suggested thatit belonged to an upright bipedal hominid who possessed a plantigrade propulsive gait (Day &Napier, Nature, Lond. 211, 1966).

Further studies on this bone, and comparative series of bones, have included canonicalvariate analysis and calculation of the D2 Generalized Morphological Distance Coefficient ofMahalanobis. Nine functionally significant characters were examined in a total of 122 bonesdrawn from populations of modern man, gorilla and chimpanzee.The results entirely confirm the previous morphological assessment and suggest that the

functional affinities of the Olduvai hominid toe bone lie with those of plantigrade bipedal manrather than with those of the pronograde quadrupedal gorilla or chimpanzee.

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Great Britain and Ireland 839The discovery of a bone with these remarkable characters, in East African Upper Villa-

franchian deposits, adds considerable weight to the view that striding heel-toe bipedalism ofhuman variety was the gait employed by the early hominids evolving in East Africa during theLower Pleistocene.

Demonstration of the collagen pattern in young articular cartilage.By M. A. MACCONAILL, University College, Cork

It is virtually impossible to demonstrate satisfactorily the pattern of collagen in young, e.g.post-natal, articular cartilage by methods that show it well in older material, even when polarizedlight is used. A series of experiments has provided a solution to this problem. 8 #rm sections onthe slide are oxidized by potassium permanganate solution (1: 1000), frequently renewed, for 2 h.They are then stained for 30 min with haplofavioxanthic acid (chromosome red) and mounted,preferably in a plastic mounting medium. They are examined in elliptically polarized light,using the compound polarizer type of microscope.The picture seen is in every way similar to that of mature cartilage with one exception: there

is, apparently, a marked decrease of the chondrousia (mucopolysaccharide and keratosulphate)as the amount of collagen increases with age.

Changes in the fine structure of the epithelium lining the urinary bladder which accompanyhyperkeratinization induced by vitamin A deficiency. By R. M. HICKS, MiddlesexHospital Medical School. (Figs. 3-5)

Rat transitional epithelium, which lines the urinary bladder and ureters, may be regarded asa keratinizing epithelium in which cornification does not normally occur (Hicks, J. Cell Biol. 28,1966). However, animals maintained on a vitamin A-deficient diet develop a characteristicpathology in which various epithelia, including that of the bladder, are replaced by cornifiedcells. The transitional epithelium, which is normally composed of three layers each one cellthick, becomes first hyperplastic (Fig. 3) and then, after ten to fifteen weeks on the diet, trans-forms into a stratified, cornified epithelium (Fig. 4) which looks remarkably like that of normalskin. It consists of a stratum basale (b) of germinal cells, an intermediate layer (i) several cellsthick, then a stratum granulosum (g) of spindle-shaped cells containing keratohyalin-likegranules, and finally a cornified layer (c). The keratohyalin-like granules of the stratum granu-losum appear in the electron microscope as large irregular shaped, dark bodies. (d, Fig. 5).The nature and formation of these granules in the hyperkeratotic bladder have been investi-

gated, and it has been shown by cytochemical staining methods that, like the keratohyalingranules of skin, they contain ribose-nucleic acid but do not have a high concentration ofdisulphide groups. Examination with the electron microscope of hyperkeratotic bladders hasshown that the keratohyalin-like granules, or dark bodies, are derived from large aggregates ofribosomes. In this tissue, the keratohyalin-like granules are the source of the dense, homogeneousmatrix (m) of the cornified layer (Fig. 5), and there is no evidence to suggest that they are inany way associated with the formation or aggregation of tonofilaments.

The fate of intramuscular homografts of thyroid gland in rats. By D. H. PARKERand R. J. SCOTHORNE, University of Newcastle upon Tyne

Fragments of thyroid gland from fifty-nine Glaxo hooded-rat donors were grafted to a rawbed in gastrocnemius muscle of Wistar albino recipients. Grafts were recovered at intervalsfrom 9 to 42 d and studied histologically. All grafts recovered at 9 d were heavily infiltrated withsmall lymphocytes. However, grafts removed at 2, 3, 4 and 6 weeks varied in appearance: somewere completely destroyed while others showed histologically healthy thyroid cells despiteheavy lymphocytic infiltration. A third group (which included some of the 42 day grafts) showedapparently normal thyroid follicles, with little or no lymphocytic infiltration. In all, about 70 %of grafts recovered at 42 d showed some histologically healthy thyroid epithelial cells.

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Proceedings of the Anatomical Society of

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840

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Great Britain and IrelandNo fully satisfactory explanation can at present be offered for the prolonged survival of a not

insignificant number of adult thyroid homografts. The degree of genetic dissimilarity between thenon-inbred strains used is shown by prompt rejection of full thickness orthotopic grafts ofhooded skin to albino recipients (mean = 11 d; range 9-16 d). That the intramuscular graftsite used for the adult thyroid grafts allows both the development and the expression of thehomograft response is shown by the fact that blocks of tissue (including thyroid gland) fromthe necks of 141-151 d hooded-rat embryos were all destroyed, by a typical homograft response,within 28 d of intramuscular grafting to adult albino recipients.

Uterine lymphatics in the rabbit. By J. M. McLEAN and R. J. SCOTHORNE,University of Newcastle upon Tyne

Many explanations have been offered for the fact that the conceptus does not sufferrejection as a homograft. The present consensus is that the foetus is protected by a non-antigenicbarrier of trophoblast, and there is little support for the idea that the uterus may be a 'privilegedsite'.

Because lymphatics (rather than blood vessels) are known to be the portal of entry of antigensfrom orthotopic skin homografts (and are presumed to be so in the case of other tissues) it isimportant to have detailed information about the lymphatics of the uterus and particularly ofthe endometrium. Non-pregnant, pseudo-pregnant and pregnant New Zealand white rabbitswere used. Uterine blood vessels were injected by successive perfusion with potassium dichromateand lead acetate solutions. The lymphatics were then injected with dilute indian ink, usinga 30-gauge needle. Injection was most commonly made into the subperitoneal or intramuscularplexuses, in the hope of filling endometrial lymphatics by retrograde flow. In a few cases theuterus was opened and stab injections made into the superficial part of the endometrium.Blood vascular and lymphatic patterns were studied in cleared free-hand or frozen sections andin serial paraffin sections. Prior injection of the blood vessels prevented confusion betweenblood and lymphatic capillaries. While the endometrium showed a very rich blood capillaryplexus, uterine lymphatic vessels were largely confined to a subperitoneal plexus, a rich intra-muscular plexus and circumferential channels lying between the endometrium and the myo-metrium. Radially arranged vessels linked the three groups of lymphatics. Within the endo-metrium itself, however, injected lymphatics were rarely found, and these few were confined tothe outermost one-fifth of the endometrium. These findings were discussed in relation to theimmunological problem of pregnancy.

Studies on the transitional cell: I. Morphology of the fixed and living cell.By C. RoSSE and J. M. YOFFEY, University of Bristol

Experimental evidence from a number of different sources points to the transitional cell asthe haemopoietic stem cell in bone marrow. To clarify a number of its morphological features,on which agreement has not yet been reached, we studied the bone marrow of fifteen normaland twenty-three sublethally irradiated (150 r) guinea-pigs. Fixed smears were stained withMacNeal's tetrachrome stain, and cells were also studied in the living state, stained supravitallywith neutral red and pinacyanol. The supravital preparations were examined under bright fieldillumination and phase contrast at 38 'C.

In fixed smears numerous intermediate stages were found between the pachychromatic smalllymphocyte and the typical large leptochromatic transitional cell. These were intermediate notonly in cell size but also in appearance of nuclear chromatin. All degrees of clumping of thenuclear chromatin have been found among lymphoid cells.These morphological features were confirmed in the living cells. In small lymphocytes a

prominent nucleolus and coarse clumps of nuclear chromatin were readily identified. In typicaltransitional cells the nuclear chromatin appeared uniformly dispersed and the nucleolus wasabsent. Numerous cells were found in which all intermediate degrees of nuclear chromatinclumping were evident. In a number of cells, which appeared uniformly leptochromatic underbright field illumination, phase contrast revealed condensations of chromatin, particularlybeneath the nuclear membrane.

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Proceedings of the Anatomical Society ofThese findings provide evidence that the appearances first noted in fixed cells are not artefacts,

but really exist. Furthermore, they make it difficult to place the small lymphocyte and thetransitional cell into two sharply separated groups.

It was found that in fixed smears transitional cells could be classified into three groups interms of different degrees of cytoplasmic basophilia. One group possessed a deeply basophiliccytoplasm, comparable to that of the proerythroblast, whereas cells in the second group hada pale cytoplasm, similar to that of the myeloblast. Cells in the third group possessed allintermediate degrees of cytoplasmic basophilia.

Experimental observations on the globule leucocyte relating it to the immune responseagainst Nippostrongylus brasiliensis. By P. WHUR, University of Glasgow

Each generation of the rat nematode N. brasiliensis is abruptly ended by the expulsion of theadult worm population from the small intestine at self-cure. Self-cure is an immune phenomenon,the timing of which is altered by changing the immunological status of the rat. In rats renderedhyperimmune by repeated infections with N. brasiliensis this response occurs about a weekearlier than in non-immune rats subjected to a single primary infection. This fact has beenutilized in suitably designed experiments to determine the relationship of globule leucocytesto the immune response. These findings, together with preliminary results of observations onthe global leucocyte response in immunologically impaired animals, were used to suggest anorigin and functional significance for these distinctive cells.

A preliminary report on answers to a questionnaire on the embalming of cadavera fordissection purposes. By G. IRVINE, University College, Cardiff

The answers to a questionnaire sent out on 1 November 1966 to all schools of anatomy inthe British Isles are being collated, and in the first instance a statistical resume has been made.The results of this are given with reference to the circumstances of cadaver storage (containers,wet/dry conditions, temperature); the technique of injections (routes, preliminary washing out,quantities of embalming fluid employed, colour-injections, variations with the nature of thecadaver); the remarkable variety of embalming fluids used; and the effectiveness of the variousmethods.

The foetal larynx. By BERNARD TOWERS, University of CambridgeIt has recently been shown (Towers, Adams & Desilets, Anat. Rec. 154, 1966) that in the lamb,

because of the intranarial position of the apex of the epiglottis, the foetal larynx opens into thenasopharynx. The opening is held shut in utero by a sling of muscle that pulls the arytenoidcartilages against the back of the epiglottis. This sphincter mechanism relaxes periodically toallow for escape of the complex fluid that is formed in the foetal lung. The anatomy of theregion was illustrated by means of serial sections, and was shown that there is a clear distinctionbetween the mucous and serous glands of the future airway and those of the lateral food-channels.From anatomical considerations it can be said that the inhalation of oral mucus by the foetusor newborn is highly unlikely.

It is not generally recognized that the same relationship of epiglottis to soft palate normallyholds (but with increased mobility of the parts) in the human foetus and neonate. In thecontroversy on this topic in 1888-89 (J. Anat. Physiol. 23) the opinion of Symington finallyprevailed: his authority established the teaching still found in textbooks and in the currentliterature, namely that the human foetal larynx, though placed at a more cranial level than inthe adult, always or normally lies with the epiglottis below the soft palate. The alternativehypothesis of Howes (J. Anat. Physiol. 23, 1889, and Proceedings passim) was soundly based, and

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Great Britain and Ireland 843is reasserted with supporting evidence. After Symington's communication to the Society,Bland-Sutton and Macalister withdrew the support they had previously given to Howes. Hadthey not done so, some aspects of respiratory physiology and pathology in the newborn wouldnot, perhaps, be as confused as they currently are.

The innervation of the tetrapod limb. By ARTHUR HUGHES and MARTIN PRESTIGE,University of Bristol

Evidence was reviewed on how the tetrapod limb is innervated during development. Con-clusions largely based on observations in Anura are as follows:

(1) The anatomical pattern of the nerves is established in the early limb bud by the ingrowthof relatively few fibres. The nerves are not formed in relation to other structures; their develop-ment is part of the primary morphogenetic processes within the limb.

(2) Subsequent differentiation of ventral horn cells continuously sends into the limb freshaxons which are guided by the primary fibres, soon greatly outnumbered by new arrivals. In thedifferentiation of the ventral horn, the cranial half is in advance of the caudal. Motility of thelimb segments develops in a proximo-distal order. There is evidence that these facts are related,and that thigh muscles are at first supplied by relatively maturer and more cranial neuronesthan those which innervate more distal segments.

(3) This rough linear order of development of centre and periphery results in many transientnerve-muscle contacts which do not conform to the final pattern of relationships. Stimulationof developing lumbar nerves in Xenopus tadpoles at first evokes a wide range of movementswhich gradually become narrowed to the final pattern of response.

Cell and fibre selection take place centrally and peripherally. Within the ventral horn, thereis turnover of cells, at its height when limb movement is most rapidly developing. Furthermore,the numbers of fibres in peripheral nerves decrease during later stages of development. InEleutherodactylus this decrease is related to loss of motor innervation in supernumerary graftedlimbs.The development of functional nerve-muscle relationships in the limb proceeds in stages, and

as with other development processes, is epigenetic.

Multinuclear nerve cells in the autonomic ganglia of the human prostate and seminalvesicle. By P. BACSICH and A. YOUNG, University of Glasgow. (Figs. 6, 7)

While multinuclear nerve cells are not uncommon in the autonomic ganglia of youngerindividuals, we were surprised to realize that many of the ganglia of the plexuses around theprostate and seminal vesicle contained between 10-20 % multinuclear cells regardless of age.In our own investigation nerve cells with up to seven nuclei were encountered (Fig. 6), whileearlier workers (Watzka, Anat. Anz. 66, 1928; Beaton, Holmes & Windle, Anat. Rec. 75, 1939)-whose results remained either unappreciated or forgotten-reported nerve cells in the sameorgans with up to fourteen nuclei. Penitschka (Anat. Anz. 66, 1929) observed comparableconditions in the human utero-cervical ganglia.On closer examination the autonomic ganglia of the above two organs seem to fall into two

distinct groups (Fig. 7): a smaller number of magno-cellular ganglia with large slightly baso-philic nerve cells which are exclusively mononuclear, and a larger number of parvo-cellularganglia with smaller strongly basophilic nerve cells many of which are multinuclear. Gangliaof the latter group also appear to have a distinctly richer blood-supply.At the present stage of our investigation it is impossible to give a satisfactory interpretation

of our findings. It may be that the multinuclearity of the autonomic nerve cells associated withthe male and female reproductive organs has a definite functional significance and one mightconsider the possibility that the parvo-cellular ganglia belong to the neuro-secretory system.On the other hand, it may well be that, contrary to orthodox views, the two types of gangliafound in the pelvic plexuses represent separate sympathetic and parasympathetic relay stations.

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A. _ _ _~~~~~~~~~~~~~~~~~~~~~~.....

.... B.:... .. .. ..s.a..

Fig. 6

Fig. 7

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Confirmation of the sympathetic adrenergic nature of paravascular axons in the catpancreas. An autoradiographic study. By T. L. B. SPRIGGS, J. D. LEVER and J. D. P.GRAHAM, University of Wales. (Fig. 8)

Earlier studies by means of a formal-fluorescence technique (Spriggs et al. Stain Technol. 41,1966) have indicated the presence of a paravascular fluorescent nerve plexus in the cat pancreaswhich could not be visualized following reserpine treatment nor after bilateral coeliac ganglion-ectomy. The adrenergic nature of this plexus was therefore inferred.

In the present study the fact that noradrenaline is actively transported into adrenergic nerveshas been utilized to confirm the adrenergic identity of paravascular axons in the cat pancreas.

Tritium-labelled DL-noradrenaline (83 gg/kg_ 560 puc/kg) was infused into the thoracicaorta of cats during 20 min. Specimens of pancreas were removed before commencing theinfusion and at various times during and after the infusion. The specimens were fixed for2 h in 1 % 0s04 (Palade) and embedded in Araldite. Ribbons of silver sections were mountedon collodion-coated slide, lead-stained and dipped in Gevaert NUC 307 photographic emulsion.The autoradiographs were stored in the dark for 2 months before being developed and examinedwith the electron microscope. Micrographs were taken of axons in juxtaposition to arteriolesor capillaries. The silver grain concentration was significantly higher (P < 0 001) in relation toaxons (A, Fig. 8) than over the field at large, a finding which suggests an uptake of radioactivenoradrenaline by, and thus the adrenergic identity of, these paravascular nerves.

Evidence of degeneration of paravascular axons in cat pancreas has been observed with theelectron microscope following bilateral coeliac ganglionectomy, thereby furnishing additionalsupport for the sympathetic adrenergic nature of these axons.

The relationship between the cerebral cortex and the pontine grey in the rabbit.By G. A. ABDEL KADER, University College London. (Fig. 9)

The Nauta & Gygax method was used to study the organization of the cortico-pontineprojection following cortical lesions in twenty-four adult albino rabbits. The results show

'-.

Fig. 8

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Proceedings of the Anatomical Society ofa wider neocortical origin of this projection (at least in the rabbit) than has been supposed, andthat considerable contributions to this projection arise from the limbic cortical structures (theposterior and anterior limbic cortices through the cerebral peduncle 'Pc', and the hippocampusthrough the fornix column 'fc') and also from a subcortical limbic structure (the mamillarycomplex 'mb' through the mamillo-tegmental tract 'mtt').

Fig. 9 illustrates the topographical relationship between the cortical origin (cerebral cortexshown in thick lines) and a superimposed cross-section of the ipsilateral pontine grey (thin line).

Dorsal view

Fig. 9

The interrupted lines roughly delimit cortical areas projecting to the superimposed pontineregions defined in the diagram. It is clear that the medially situated cortical (and subcorticallimbic) structures retain a medial relation within the pontine grey. The rostral part of the cortex(which includes the motor cortex) projects to the dorsal levels of the pontine grey (i.e. thereticular tegmental, the dorsal, and dorsolateral nuclei and the adjacent strip of the paramediannucleus), with the possibility that the mediolateral (leg-arm-face) sequence in the cortex ispreserved within this projection area in the pons. The caudal part of the cortex (including therostral parts of the visual and probably the auditory cortex) projects to the ventral and theperipheral lateral parts of the pontine grey. The rest of the cortical field projects to the peduncularand adjacent parts of the lateral grey.

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The long somato-sensory pathways to the rat thalamus. By K. E. WEBSTERand R. D. LUND, University College London. (Fig. 10)

The distribution of degenerating axons in the rat diencephalon has been studied by the methodsof Nauta & Gygax (Stain Technol. 29, 1954) and Guillery, Shirra & Webster (Stain Technol. 36,1961) following lesions in the gracile and cuneate nuclei, spinal cord and trigeminal nuclei.Most of the findings are summarized in Fig. 10. The caudal parts of the dorsal column nucleiproject to the contralateral ventrobasal complex (VB) in a topographically organized manner-and in an unorganized way to the medial geniculate complex pars medialis. The rostral parts

trigeminal complex Q rostral n. cuneotus ID rostra n. gracislis ED spinothalarmic tract

i rostral cord (?n.cervicclislat.)

Fig. 10

of these nuclei project to more widespread thalamic areas, namely the ventrobasal complex(in a way apparently indistinguishable from that of the caudal parts of the nuclei), the medialgeniculate complex pars medialis, the posterolateral complex pars lateralis (LP 1), the nucleuspretectalis anterior pars ventralis, and the zona incerta (ZI). The spinothalamic tract projectsto the rostral one third only of the ventrobasal complex (VB), to the medial geniculate complexpars medialis, the posterolateral complex pars lateralis (LP 1), the nucleus centralis lateralis(Cl), the nucleus medialis dorsalis pars lateralis (DM), and the nucleus pretectalis anterior parsventralis. The two most rostral cervical segments have, in addition, a projection to a localizedregion of the ventrobasal complex (VB) which may arise from the lateral cervical nucleus; anda small projection to the parafascicular-centrum medianum complex (Pf). The trigeminal nucleiproject to the ventrobasal complex (the contribution from the pars caudalis being more restrictedthan that from the rest of the trigeminal nuclei), the medial geniculate complex pars medialisand the posterolateral complex pars lateralis. No long ascending fibres have been seen to endin the nucleus reticularis thalami (R) or in the rodent homologue of the posterior group. Theresults were discussed in relation to recent functional studies.

53 Anat. IOI