GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

25, 126- 146 (1975)

The Pituitary Gland of the Coelacanth Latimeria chalumnae Smith MICHAEL Department

D. LAGIOS

of Pathology, Children’s Hospital of San Francisco, San Francisco, California 94119

Received August 28, 1974 Pituitary structure in the coelacanth Latimeria, the surviving member of the Crossopterygii, demonstrates features both shared and unique amongst the bony fishes. The neurohypophysis contains three areas of specialized neural contact. The anterior neurohypophysis comprises both a neurohemal organ-a median eminence -and an area of more direct, interdigitate contact with the pars distalis reminiscent of Amia. The predominant axon type of the median eminence contains a small monaminergic-type, dense-cored vesicle of 950 8. diameter. A neurointermediate lobe complex consists of branching tubular processes of the posterior neurohypophysis and follicles of the pars intermedia. The former contain peptidergic-type axons with large dense-cored vesicles of 1960 8, diameter which stain strongly as elementary neurosecretory granules (Knowles Type A). The saccus vasculosus is incompletely separated from the peptidergic posterior neurohypophysis. The tripartite pars distalis shows a more marked tendency toward compartmentalization relative to the lower Actinopterygii and the Dipnoi. Two major divisions of the pars distalis are evident on the basis of vascularization. The more orthodox proximal division is closely associated with the neurointermediate lobe and the portal vessels which appear to represent the major blood supply to this area. It comprises a purely acidophilic dorsal lobe (orangeophils and erythrosinophils), and a posterior lobe of mixed-cell type (orangeophils and basophils) which has a limited area of direct interdigitate neurohypophysial contact. The elongate extension of the pituitary so peculiar to the coelacanth comprises the rostra1 division which is connected only by a tubular hypophysial cavity to the remainder of the distalis. This virtually separated lobe has a substantial direct arterial supply derived from the internal carotids and contains relatively few chromophilic cells of basophil type in this immature female. The organization of the neurohypophysis in Latimeria is similar to that of other jawed fishes, particularly some relic actinopterygians. In contrast, the tripartite division of the pars distalis, the histologically separated rostra1 division with its direct arterial supply, basophil cell type, and close association with the carotid anastomosis, are unique features among the Osteichthyes, but are strongly reminiscent of the elasmobranch ventral lobe. Pituitary organization in Latimeria contrasts markedly with that of the lungfishes. The significance of this disparity in terms of the relationship of the two Orders of the Sarcopterygii (Romer) to each other, and of the unique features of the coelacanth in terms of its position as a sister group to the Rhipidistia, is discussed.

Latimeria chalumnae, the surviving mesobenthic member of the specialized marine Coelacanthini, affords a limited but altogether unique opportunity to examine pituitary organization in what is generally regarded as an extant crossopterygian. The single coelacanth together with the small group of relic lungfishes comprise the subclass Sarcopterygii. Both groups are

characterized by separate ancient lineages which were distinct in the lower Devonian strata in which they are first evidenced. They also share a marked conservatism of form and habitat, and an enlarged genome size, of extreme proportions in the Dipnoi (Thomson, 1969; Ohno, 1969). Pituitary organization tends to be a remarkably uniform and stable feature within 126

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@ 1975 by Academic Press, Inc. of reproduction in any form reserved.

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any one of the major vertebrate groups, extant fishes or tetrapods. Although distorted at times by developmental processes, basic pituitary structure remains relatively stable compared to other morphologic features on which selection pressure can bear directly. Thus, common patterns of pituitary organization are seen within the Chondrichthyes and separate but consistent patterns within the lower Actinopterygii and the radical Teleostei. The striking morphologic conservatism of the relic lungfishes and the coelacanth suggests that their basic pituitary structure probably closely approximates that of the extinct host of primordial sarcopterygian fishes. The present work describes the pituitary gland of Latimeria chalumnae by light and electron microscopy, and compares it with that of other fishes. Particular attention is given to the implications of this latter comparison to the question of the Sarcopterygii as a valid assemblage and the Coelacanthini as a sister group to the Rhipidistia. MATERIALS

AND METHODS

The pituitary gland described in this study was obtained from an 86-cm immature female Latimeria chalumnae caught by handline at night in approximately 300 m off Iconi, Grande Comore on 22 March 1972. The specimen was dissected after observations of the living fish had been completed (Locket and Griffith, 1972). The brain and attached proximal 25 mm of the pituitary complex were dissected intact and fixed by immersion in an iso-osmolar glutaraldehyde (Lagios, 1974). The remaining more rostral 20 mm of the pituitary complex were fixed in situ within the cranium in 10% formalin, and subsequently dissected after some delay. The gross morphology of the pituitary gland was quite similar to that observed previously in a 130-cm adult male specimen (Lagios. 1972). Gross photographs and drawings were prepared of the intact fragments to aid subsequent orientation and study. The brain including the medulla oblongata, cranial nerves, and the pineal gland, were then separated from the pituitary complex and distributed to other investigators. Because of the limited nature of the material it was elected to divide the better-fixed proximal portion of the pituitary gland into thick (ca. I mm) free-hand,

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parallel frontal sections or layers, and utilize alternate layers for light and electron microscopy. In practice the layers obtained for electron microscopy were much thinner than those used for conventional study. Fragments were excised from the intact layers which could be compared with the corresponding area in the facing surface of alternate light-microscopic sections, and for precise correlation small areas excised from unmounted nitrocellulose sections were processed for electronmicroscopy permitting study of comparable areas with both methods. The rostra1 portion of the infundibulum was transversely sectioned to obtain a cross-sectional segment of the median eminence, previously identified by serial frontal sections (Lagios, 1972), for ultrastructural study. Layers used for light microscopy were embedded in nitrocellulose, serially sectioned at 6 pm, and mounted on consecutively numbered slides by a method used previously (Lagios, 1968). Alternate sections were stained routinely with periodic acidSchiff-Orange G (PASOG) with a Harris hematoxylin nuclear stain preceding the Orange G. The remaining sections were stained variously with performic acid-Alcian Blue 8GS (PFAAB) alone and in combination with subsequent PASOG, Luxol Fast Blue-PASOG, acid Alcian Blue (pH 0.2) without prior oxidation, and Herlant’s (1960) tetrachrome with erythrosine-Orange G. Additional slides were stained with PAS followed by a modification of the erythrosine-Orange G differential step for eta acidophils employed by Herlant, and by the lead-hematoxylin method of MacConnaill (1947). Alternate serially sectioned stained slides were photographed on Panchromatic film, enlarged on Kodak fine-grain 8 X IO-in. positive film, and developed to provide superimposable positive transparencies. These were used to study the relationships of the neuro- and adenohypophyses. Tracings of the enlargements were prepared as line drawings. A comparison was made of the present incomplete serial series with the complete serially sectioned pituitary gland from the adult male. Tissue for electron microscopy was postfixed in osmium tetroxide, embedded in Spurr and Epon, sectioned at 1000 K with a Porter-Blum MT2 ultramicrotome with diamond knives. Sections were stained with uranyl acetate and lead citrate on uncoated copper grids and viewed with a Zeiss EM 9 S2 electron microscope. The most distal 20-mm pituitary fragment which had been fixed in situ showed evidence of considerable postmortem autolysis indicated by the loss of stain reactivity, cytoplasmic vacuolation, and disruption of organelles noted in small portions embedded for electron microscopy. The remainder of this distal fragment was embedded in paraffin and serially sectioned. Because of the lack of affinity for the staining procedures used on the better-fixed proximal portion.

128

MICHAEL

D. LAGIOS

A .bbreviations: A, artery; AX, axon; BM, basement membrane; C, hypophysial cavity; CA, capillar J’: E, endot1tiehal cell; EP, ependymal cell; F, follicle boundary cell; G, glial process; IN, infundibulum, IS, isle:t of rostral lobe, pars distalis; NH (NHA, NHP), neurohypophysis (anterior and posterior); PD, pars distalis; PFI AAB, performic acid-Alcian Blue procedure; PV, perivascular space; V, extension of ventricula r cavity; sv, saccus vasculosus.

F‘IG. 1. Pituitary orientation. Schematized tracing of representative frontal section through proximal pituitary Level is ventral to median eminence and includes portions of dorsal, posterior, and roscon rplex in Latimeria. trat lobes of pars distalis, PD. Ventral portion of infundibulum, NH. Ventricular cavity, V, extent ds as tuhuh u processes into the neurohypophysis (unshaded). The lateral portions of the neurohypophysis r.epresent the NHP and interdigitate with the pars intermedia, PI (light shading) to form the neurointermedi ate lobe.

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Mallory trichrome was employed largely to delineate the vascular relationships of this area. An immature 26-cm Lepidosiren paradoxa was anesthetized with quinoladine and perfused through the heart with a 1.5% glutaraldehyde in cacodylate buffer (pH 7.2, mOsm 340) containing 4% polyvinyl pyrrolidine (MW 40,000). The minute l-mm pituitary complex was divided into an anteroventral segment containing the portal vessels and pars distalis, a dorsal segment containing the neurointermediate lobe and transverse segments of the median eminence region. The tissue was submitted for electron microscopy in order to more directly compare the coelacanth material with a dipnoan which has been previously studied (Zambrano, 1972, 1973).

OBSERVATIONS

Gross morphology. The striking gross feature of the coelacanth pituitary is the peculiar elongation of the pars distalis noted by Millot and Anthony in their initial description (1955). From an orthodox position beneath the optic chiasm, a wellvascularized fibrous sheath, coextensive with the meninges, invests a greatly attenuated rostra1 extension of pituitary tissue which is intimately associated with the adventitia of the internal carotids as they enter the cranial floor through the basisphenoid in the hypophysial recess (Millot and Anthony, 1958; 1965; Lagios, 1972). The entire pituitary complex in Latimeria is anteriorly rotated so that the bulk of the neurointermediate lobe is ventral rather than posterior. On the dorsal aspect of the neurointermediate lobe, a shallow longitudinal fissure carries the portal vessels. Just anterior to the sheaf of white portal vessels, a number of small nodular excrescences bulge into the dorsal surface of the fibrovascular sheath. These correspond to the dorsal lobe of the pars distalis. More anterior still, smaller excrescences indicate the location of the discon-

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tinuous rostra1 division. In the present 86-cm immature female the rostra1 division is estimated at 4 cm in length, while in the previously dissected 130-cm adult male it was 8 cm. The gross morphology and general histology (Fig. 1) of the present and the previously described material (Lagios, 1972) differ in no significant respect. Neurohypophysis. Light Microscopy The neurohypophysis of Latimeria may be divided into an anterior neurohypophysis, comprising a median eminence and areas of more direct, interdigitated contact with the pars distalis, and a posterior represented by the neurohypophysis, neurointermediate lobe (NIL). A dorsal, unpaired, tapered, thick-walled blunt-faced portion of the anterior neurohypophysis abuts against capillaries of the primary bed and comprises the median eminence (Fig. 2). Between the bifurcation of the posterior neurohypophysis and the more dorsal (anterior) median eminence, branching processes of the anterior neurohypophysis form a limited but more direct interdigitated contact with the pars distalis (Fig. 3). These processes are morphologically similar in their relationship to the distalis to the short processes in Amia (Lagios, 1970) which similarly provide a limited, more direct teleost-like interdigitate contact with the posterior portion of the presumptive proximal pars distalis. The neurointermediate lobe complex (NIL) is a compact globose mass closely attached to the hypothalamus immediately inferior to the optic chiasm and median eminence. It consists of paired ventricular evaginations of the posterior neurohypophysis which ramify in a complex fashion

Arrow indicates portion of the posterior lobe of PD in direct contact with interdigitate portion of NHA. The posterior lobe is here separated by level of section from the bulkier dorsal lobe of PD (indicated by PD in hgure). Note the tubular extensions of the hypophyseal cavity, C, into the dorsal lobe of PD, and its rostra1 continuation as a single tubular cavity from which the more proximal and intermittent portions or islets, IS, of the rostra1 lobe of the PD develop. Scale 1 mm.

FIG. 2. Median eminence, 1 pm transverse plastic section stained with Toluidine Blue. A, segments of convoluted preportal arterioles. Primary capillary bed represented by capillaries embedded in leptomeningeal fibrous tissue plaque. Scale 0.25 mm. FIG. 3. Anterior neurohypophysial processes interdigitate with posterior lobe of pars distalis. Note adjacent pars intermedia and posterior neurohypophysial processes darkly stained with PFAAB. Nitrocellulose 6 pm section. Scale 100 Gm.

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within the follicular pars intermedia (Fig. 4). The NIL is closely invested by a thick, highly vascular fibrous extension of the leptomeninges covering the infundibulum. The saccus vasculosus is also invested by this same vascular sheath. Neurohypophysis.

Electron

Microscopy

Median eminence. The neural components of the median eminence of Latimeria differ in no significant way from the morphology of the anterior neurohypophysis in relic actinopterygians (Lagios, 1968, 1970; Hayashida and Lagios, 1969; Polenov, 1968, Polenov et al., 1972) and tetrapods. Numerous large axonal endings abut against the cerebral basement membrane which forms one border of a perivascular space (Figs. 5-6). No elaborate convolutions or interdigitations of the cerebral basement membrane are demonstrated. The axonal endings show no appreciable staining with procedures designed to demonstrate cystine linkages typical of ENS (elementary neurosecretory, type A) granules. Three classes of axonal endings are recognized by specific types of densecored vesicles; all contain identical SOOA diameter synaptic vesicles or synaptosomes. Two classes are more frequently represented and are morphologically monaminergic in type, the third is uncommon and appears to be peptidergic in type. The most frequent axon contains a class of small 800- 1100-A diameter dense-cored vesicles with a characteristically elongated, pleomorphic profile and a long axis up to 2000 A. A clear zone or halo is seen about the dense core of the vesicle. Two additional classes of dense-cored vesicles are seen in axons abutting on the perivascular space. Both are uniform and round in their configuration in contrast to the elongated pleomorphic type: an equally electron-dense, non-pleomorphic monaminergic type 1OOO- 1160 A in diameter, and a similarly uniform but somewhat less electron-dense peptidergic type 1300

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131

A in diameter. The axonal endings show a presynaptic membrane density, and occur singly and as small groups separated by conspicuous glial footplates. The perivascular space is quite thick, l-7 pm as in Pofyodon (Hayashida and Lagios, 1969) and contains large bundles of collagenous fibers, tissue eosinophils, mast cells, and melanocytes. The primary capillary bed, arranged as complex convoluted vessels or glomeruloids, lies in a thick fibrous plaque which was better appreciated in the larger but poorly preserved 130-cm adult male (Lagios, 1972). A similar plaque containing the primary capillary bed and convoluted arterioles of the portal system is seen in the polypteriformids (Kerr, 1968; Lagios, 1968) and Amia (Lagios, 1970). In this immature female the thick overlapping endothelia of primary capillaries rarely show fenestrations. Transverse sections through the infundibulum demonstrate a prominent complete ventricular surface of ependymoglial cells which have welldeveloped tripartite junctional complexes and develop occasional cilia of the 9-2 couplet configuration. However, no coronet cells (Kroenchenzellen) are present. No axons terminate on the ventricular surface, although the ependymoglia itself is innervated by monaminergic type axons both on the subventricular (basal) perikarya, and on the glial footplates bordering the median eminence. The perikarya of occasional glial cells lie within the substance of the infundibulum. Between the thick ependymoglial layer and the median eminence numerous axonal tracts are seen. The majority contain dense-cored monaminergic-type vesicles similar to those seen abutting on the perivascular space. Posterior Neurohypophysis (Pars Nervosa)

The racemose tubular processes of the posterior neurohypophysis intermingle with the follicles of the pars intermedia. They penetrate the external aspect of the

FIG. 4. Neurointermediate lobe. Tubular processes of posterior neurohypophysis interdigitate with foilicular pars intermedia. Note tubular pure ependymal processes of saccus vasculosus (arrows) at periphery of neurointermediate lobe. Nitrocellulose, 6 pm. Scale 100 pm. FIG. 5. Median eminence electron micrograph. Monaminergic-type axons, AX. interdigitate with glial footplates (arrows) on cerebral basement membrane bordering a perivascular space. The latter contains collagenous fibers and a fibrocyte (arrow). Scale 0.5 pm.

FIG. 6. Median eminence electron micrograph. Detail of axonal processes on cerebral basement membrane demonstrating two morphologic forms of small dense-cored vesicle of monaminergic type: (1) uniform; and (2) elongated. Scale 2000 A. FIG. 7. Neurointermediate lobe, l-pm plastic section stained with Toluidine Blue. Thick-walled follicles of the pars intermedia with abundant follicle boundary cells bluntly interdigitate with posterior neurohypophysial processes. Coronet cells (Kroenchenzellen) are indicated by arrows on the ependymal surface of the tubular processes. Scale 50 pm.

FIG. 8. Neurointermediate lobe electron micrograph. Pars intermedia is composed of predominant cell with large, flocculent secretion granules, and less common pyramidal cell (arrow) with dense, smaller secretion granules. Scale 5 pm. FIG. 9. Neurointermediate lobe electron micrograph. Predominant cell of pars intermedia separated from perivascular space by process of follicle boundary cell. Note that intervening perivascular space contains collagenous fibers and both adenohypophysial and neurohypophysial basement membranes. Scale 5000 A.

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pars intermedia as well as ramifying from within. All retain a central ependymoglial core which represents a tubular extension of the ventricular space. Axonal processes surround the ependymoglial core. The axons demonstrate a striking PFAABpositive reaction (Figs. 3, 7-9) and electron microscopy reveals large dense-cored vesicles of peptidergic type. The majority of the axonal endings terminate on the cerebral basement membrane bordering a fibrovascular space which invests the follicles of the pars intermedia. The boutons termineaux are closely invested by glial footplates. Hemisynapes (synaptoid contacts) with the basement membrane are often made by an axonal nubbin replete with synaptosomes. The nubbin extends between adjacent investing glial footplates in a manner similar to peptidergic NIL contacts in Calamoichthys. The fibrovascular space between the processes of the posterior neurohypophysis and the pars intermedia contains abundant collagenous fiber bundles and measures 0.6- 1.O pm in thickness. Great attenuation of this space with resultant apposition of the cerebral and intermedia basement membranes as seen in Calamoichthys (Lagios, 1968) and direct synaptic innervation as seen rarely in Amia (Lagios, 1970) and more commonly in teleosts (Zambrano, 1970) and tetrapods is not demonstrable. Axons associated with the fibrovascular space investing the pars intermedia contain peptide&-type vesicles of 1650 A average diameter (1500-l 800 A). Perivascular contacts, synapses terminating in close proximity to a capillary wall, are infrequently seen, and although of similar electron density and configuration, measure up to 2 100 A in diameter. The posterior neurohypophysis also contains infrequent axons with small 980 A average diameter dense-cored monaminergic type vesicles similar to the non-pleomorphic type I axons of the median eminence. These abut on glial footplates, peptidergic axonal processes and

135

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endings and also on the cerebral basement membrane, both in association with pars intermedia and vessels. Cerebral basement membrane may show convolutions in areas of monaminergic type perivascular contact in the NIL. Saccus

vascalosus

A striking feature of Latimeria is the incomplete anatomical separation of the saccus vasculosus from the posterior neurohypophysis. Both neural systems originate as tubular ependymal evaginations from the same paired primary processes of the posterior neurohypophysis, and both are closely invested by the leptomeninges. A morphologic gradation occurs between the branching neural processes comprising the NIL and a more peripheral group of pure ependymal tubules (Fig. 7). The former processes, particularly at the lateral border of the NIL, contain characteristic peptidergic type axons but their ependyma contains typical coronet cells (Kroenchenzellen) as well. Axonal processes are reduced and coronet cells increased in the more peripheral neurohypophyseal processes whose morphology is similar to the saccus vasculosus in Polyodon. In addition to the close anatomic origin and histologic gradation evident between the two tubular systems, coronet cells themselves, the hallmark of the specialized ependyma of the saccus, are more widely distributed and occur in otherwise typical posterior neurohypophysial processes in the central portion of the NIL. Hansen (197 1) has described a similarly more diffuse distribution of coronet cells in the relic actinopterygians in which they occur in the eminentia. Similarly diffuse distribution of coronet cells occurs in some elasmobranchs, Heterodontus and Triakis. However, the relic chondrostean and holostean fishes show a more discrete, anatomically separate saccus (Kerr, 1968; Lagios, 1968, 1970; Hansen, 197 1).

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MICHAEL

Pars Intermedia

The pars inter-media consists of thickwalled irregular follicles whose lumina are often compressed. Two endocrine cell types are evident (Figs. 7-8). A predominant large-granule type with flocculent (uncondensed), round 3600-A diameter granule profiles comprises approximately 80% of the cells present. A clear space between the flocculent secretion granule and the limiting membrane is not seen. These cells lie stratified up to three deep within the follicular wall. Their cytoplasm stains with lead-hematoxylin and faintly with PAS; their nuclei are large with numerous small chromocenters. A minor pyramidal cell type shows a more pronounced PAS-positive reaction, fails to stain with lead-hematoxylin, and contains numerous small 2600-A diameter electrondense secretion granules with a characteristic perigranular “halo” or clear space, and dilated cisternae of the rough-surfaced endoplasmic reticulum. Nuclear morphology is elongated although the chromatin pattern is similar to that of the major cell type. The pyramidal cells are restricted to the vascular surface of the follicle and comprise approximately 5% of cells present within the follicle. Their appearance and distribution are similar to the pyramidal cells of the teleost neurointermediate lobe (Lagios, 1965; Ball and Baker, 1969). Follicle boundary cells are a prominent additional non-endocrine component of the pars intermedia and constitute approximately 15% of the epithelial cells in the NIL. Like their counterparts in the chondrostean fishes (Lagios, 1973), follicle boundary cells in Latimeria form a complete epithelial barrier around colloid-filled follicles. No endocrine cells abut on the follicular lumen as in Amia (Lagios, 1973) and in teleosts (McKeown and Leatherland, 1973). Elaborate tripartite junctional complexes lock adjacent cell margins about the lumen. These cells envelop en-

D. LAGIOS

docrine cells in thin cytoplasmic processes and extend to the vascular surface where they characteristically form large epithelial foot processes analogous to glial footplates. The foot processes of the follicle boundary cells lie between the endocrine cells and the basement membrane of the intermedia and thereby tend to limit contact of the latter cell type with the perivascular space. Follicle boundary cell epithelium is not limited to the pars intermedia but occurs as a pervasive system lining follicles in the pars distalis and surfacing the hypophysial cleft and its rostra1 tubular extension. A similar distribution occurs in the Chondrostei (Lagios, 1973). Numerous small juxta-luminal PAS-positive vesicles in follicle boundary cells are seen, particularly in the pars distalis. These contain an electron-dense material. The vesicles occasionally can be seen to fuse with the luminal cell membrane, releasing their product in a manner resembling exocytosis. Pars Distalis Vascufarization. Although vascular injection studies were not possible in the limited material available, some preliminary interpretation of vascular patterns within the pars distalis is possible. Based on these differences, the pars distalis may be divided into proximal and rostra1 divisions but are not meant to correspond to the PPD (proxima1 pars distalis) and RPD (rostra1 pars distalis) of teleost fishes. The more orthodox portion of the pars distalis comprises the proximal division which forms a relatively compact mass dorsal to the hypophysial cleft which separates it from the more ventral NIL. A similar arrangement is seen in the serially sectioned but poorly fixed adult male (Lagios, 1972) and in the sagitally sectioned figure of Millot and Anthony (I 965). The proximal division lies directly anterior to the sheaf of recognizable portal vessels

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which lie in the dorsal longitudinal depression in the NIL. These vessels appear .to represent the major, if not the exclusive, blood supply to the proximal division. The rostra1 division comprises the markedly elongated extension of the pars distalis so peculiar to the coelacanth pituitary (Millot and Anthony, 1955; Lagios, 1972). The rostra1 division is virtually separated from the proximal by the fibrous vascular sheath which surrounds the NIL and extends anteriorly into the hypophysial recess. A striking feature of the rostra1 division evident in the serially sectioned, poorly fixed material available from the present 1972 specimen, is a substantial independent vascularization. Small muscular arteries derive from the internal carotids at an oblique angle and run retrograde parallel to the parent vessel before arborizing

137

into arterioles and then merging with the capillaries surrounding the follicles of the rostra1 division (Figs. 10, 11). Review of the previously studied adult male (Lagios, 1972) reveals similar arterial ramification within the midportion of the rostra1 extension. Histology. The entire pars distalis, as well as the pars intermedia, demonstrate a follicular organization, and follicle boundary cells are a conspicuous component of the follicles. All parts of the pars distalis preserve an association with the persistent hypophysial cleft, which is expanded in the proximal division and tubular in the rostra1 division. Tubular evaginations of the hypophyseal cleft in the proximal division are associated with acidophils of the dorsal lobe in a pattern similar to that described in Acipenser (Kerr, 1949; Hansen, .

FIG. 10. Rostra1 lobe of pars distalis, paraffin carotids, A, surrounded by autolyzed endocrine branches of the internal carotids which originate vessel. Origin of the internal carotids (proximal)

section. Longitudinal segments of major branches of internal tissue of rostral lobe, PD. Arrows indicate takeoffs of two at an obtuse angle and mn retrograde paralleling the parent is to the left in the figure. Scale 0.25 qn.

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MICHAEL

D. LAGIOS

FIG. 11. Rostra1 lobe of the pars distalis. Transverse section through the midportion of the rostra1 lobe of the pituitarv revealing- autolvzed _ endocrine tissue, PD, and ramifying arterial vessels. A, originating from a larger radicle of the internal carotids. Scale 0.25 pm.

197 1). Segregation of cell types within the pars distalis is pronounced in this immature female. Three histologically distinct areas (lobes) are discerned within the pars distalis. Two lobes are apparent within the proximal division, the component which receives a portal vascular supply. A dorsal lobe is composed of follicles and irregular trabeculae in which two acidophil cell types are sharply segregated. The more anterior portion of the dumbbell-shaped lobe forms a circumscribed mass which can be appreciated in the gross specimen as a nodule on the dorsal aspect of the leptomeningeal sheath. It contains large columnar erythrosinophils; the more posterior portion contains typical orangeophils (Table 1). Although some intermixture of cell types occurs in the adjacent posterior lobe, the more superior aspect of the dorsal lobe contains only two acidophil

cell types and follicle boundary cells. These features suggest homology with the rostra1 pars distalis of teleosts. A posterior lobe lies inferior and posterior to the dorsal but is not separated by fibrous tissue. It consists of longitudinally oriented trabeculae paralleling the portal vessels and contains a mixed population of orangeophils and PAS-positive cells. The posterior lobe merges with the NIL laterally and directly interdigitates with a limited portion of the anterior (non-peptidergic) neurohypophysis in its extreme posterior end, features which are reminiscent of the PPD of teleosts. The rostra1 division is virtually separated by fibrous tissue from the remainder of the pars distalis. Intermittent islets of this division merge into the more continuous portion which terminates on the carotid anastomosis in the hypophysial

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TABLE

1

CELL TYPES OF THE PARS DISTALIS~

Histochemistry Cell type

Distribution

E

OrG

PAS

PbH

DL

PL

RL

Granule configuration

Erythrosinophil

+

+

+

+

+

-

-

Orangeophil

-

+

+

-

+

+

-

Basophil I

-

-

+

-

-

+

-

Basophil II

-

-

+

-

-

-

+

Large, round, 2750-A diam (2180-3880) Large, round, 2160-A diam (1580-2690) Small, uniform, 1400-A diam (920- 1870) Small, pleomorphic, 1730-A diam (1090-2500)

u Abbreviations: E, erythrosine; OrG, orange G; PAS, periodic acid-S&ii, PbH, lead-hematoxylin; DL, PL, RL, dorsal, posterior and rostra1 lobes, respectively; + , positive reaction; *, weak reaction; - , negative reaction. Diameter given represents average, ranges given in parentheses. Data for basophil I is preliminary; fixation was not entirely satisfactory for ultrastructure in this area.

FIG. 12. Islet (intermittent proximal portion of rostral lobe) of pars distalis, l-pm plastic section stained with Toluidine Blue. A portion of a large follicle borders a capillary, CA. Darkly stained, granulated endocrine cells border the vascular surface of the follicle wall, while a continuous layer of follicle boundary cells borders the luminal surface. Note the debris within the follicular lumen. Scale 50 Frn.

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MICHAEL

D. LAGIOS

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recess. Relatively few chromophilic cells, the majority of basophil cell type in the tissue available, comprise the rostra1 division in this immature specimen (Figs. 12, 13). DISCUSSION Neurohypophysis

The median eminence of Latimeria does not differ significantly in its organization or ultrastructural features from those described in relic actinopterygians and tetrapods such as Necturus (Lagios, 1968, 1970; Hayashida and Lagios, 1969; Hansen, 197 1). In all primitive ray-finned fishes and in Latimeria a characteristic small, dense-cored vesicle, 900- 1000 8, in diameter, is seen in the predominant type of axonal process abutting on the perivascular space of the median eminence, the neurohemal contact region of the anterior neurohypophysis (Table 1). These anterior neurohypophysial axons share a similar histochemistry and ultrastructure, and have been shown to be monaminergic by appropriate fluorescent studies. Monaminergic-type (PFABB-negative) axons are the predominant or the exclusive (Amia) axon type found in this location, and an origin in lateral tuberal nuclei for these axons has been demonstrated in both relic actinopterygians (Sathyanesan and Chavin, 1967) and teleosts (Zambrano, 1970). Axons derived from the preoptic nucleus and its hypothalamo-hypophysial tract, in contrast, stain positively with PFAAB and the Gomori-aldehyde procedure. They have a consistent ultrastructure with mean dense-cored vesicle diameter in excess of 1350 A, and are variously termed

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“elementary neurosecretory granules,” “Type A granules” (Knowles and Vollrath, 1966), or “peptidergic axons.” They can be categorized in a number of species into two morphologic classes based on densecored vesicle size, both associated with the neurointermediate lobe. One class is associated with axons making both indirect and synaptic (teleosts) contact with the pars intermedia cell. A second class is associated with a variably developed neurovascular contact in the NIL. Thus, despite their disparate phylogenetic histories, most bony fishes and tetrapods share a monaminergic innervation in the anterior neurohypophysis, either developed as a neurohemal organ or in teleosts, as a homologous but more direct neurohypophysial interdigitate and/or synaptic contact with the pars distalis. In seeming contrast to primitive actinopterygians and tetrapods, Lepidosiren is remarkable for its apparent peptidergic anterior neurovascular contact zone, a structure previously considered homologous to the median eminence (Wingstrand, 1956), and its direct innervation of the pars distalis (Zambrano, 1972, 1973). In this dipnoan, monaminergic axons derived from tuberal nuclei are reported to extend directly into pars distalis where they lie apposed to the plasma membrane of endocrine cells. Presynaptic membrane specialization is apparently lacking, but there is no intervening basement membrane or collagenous intervascular space. If the peculiar neurohypophyseal organization in Lepidosiren is representative of the Dipnoi, it would be in contrast to the basic gnathostome pattern of a monaminergic rather than peptidergic median eminence and absent direct innervation of the pars

FIG. 13. Islet of pars distalis, electron micrograph. A basophil endocrine cell replete with small, dense pleomorphic secretory granules forms a small part of the follicle wall of the rostra1 lobe islet. The majority of the epithelial cells in this immature female comprise follicle boundary cells which border the luminal surface. Their apical cytoplasm contains numerous large periluminal vesicles with an electron-dense flocculent content. Scale 1 pm.

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distalis.

Our limited observation on indicates a dense-cored vesicle of 1 I 10 A average diameter (range 940-1400 A) and synaptosomes of 540-A diameter. These figures are intermediate (Table 2) compared to other fishes, but tend to support their purported peptidergic nature (Zambrano, 1973). Generally the presence of a median eminence appears to exclude any significant direct innervation of the pars distalis, although a meagerly developed, nonsynaptic accessory innervation occurs in Amia and in Latimeria. Teleosts, a recent group dating from the Jurassic, are the only vertebrate group known in which synaptic innervation of the pars distalis occurs, and apparently it represents a secondary innervation which supplants the median eminence in that assemblage. Studies of apparent portal systems reported in the teleosts Salvelinus and Heteropneustes are contradictory (Honma and Tamura, 1967; Hill and Henderson, 1968; Sundararaj and Viswanthan, 197 I ; Sathyanesan and Haider, 1971). A median eminence in Heteropneustes could not be Lepidosiren

COMPARATIVE IN RELIC

TABLE 2 DENSE-CORED VESICLE DIAMETERS ACTINOFTERYGIANS AND Latimeria” ME

Calamoichthys Acipenser Pofydon Amia Latimeria Lepidosiren

945 (910-1060) 842 (765-920) 9% (920-1055) 890 (800-I 100) 950 (900-1100) 1110 (940-1406)

PI

PN

1755 (1220-2290)

1370 (1060-1670)

1450 (1370-1530) 1385 (132&1450) 1570 (1240-2010) 1650 (1580-1800) 1650/950

SYN 450 (460-610) (460-580) (445-490)

2100

504

1650195CF’

ml

“Data summarize previous work of author (Lag&, 1968; 1970; Hayashida and Lagios, 1%9) and that of Zambrano* (1972a. 1972b). Diameters are given in A; average diameter and ranges are given (in parentheses). ME, median eminence; PI. pars intermedia associated innervation; PN, “pars nervosa”-peptidergic neurovascular contacts in the neurointermediate lobe; SYN, synaptosomes. A single peptidergic dense-cored vesicle type is indicated where differences were not appreciated, as in Acipensar, Polyodon. and Amia. a Indicates data from Zambrano.

D. LAGIOS

confirmed ultrastructurally by Dr. Sundararaj (1974, personal communication). Pars Distalis

The pars distalis of Latimeria, unlike that of other bony fishes, is represented by two physically separated components. These separate components appear to reflect distinctly different vascularizations (Fig. 14). The proximal division occupies an orthodox position in close association with the NIL. The sheaf of portal vessels runs anteriorly and terminates about the follicles of this division in a manner not unlike that seen in chondrostean fishes. The rostra1 division, in contrast, is tenuously connected to the remainder of the pars distalis only by the tubular hypophysial cavity and comprises intermittent islets of tissue which merge distally into a more solid, follicular mass surrounding the carotids and their anastomosis in the hypophysial recess. Arterial vessels branch from the larger internal carotids, run retrograde paralleling the parent vessel, and then ramify to supply the capillaries of the rostra1 division. Thus, there is a substantial direct arterial supply to the rostra1 division of the pars distalis. Although it is the rostra1 pars distalis in elopidormid and clupeiformid teleosts that retains an association with the buccohypophysial canal (Olsson, 1968), the rostra1 division in Latimeria does not appear to be homologous.’ Rather, the close association of this division with the internal carotids and their anastomosis in the floor of the hypophyseal recess, its independent vascularization, and at least in this immature female, the scant basophil cell type evident in the discontinuous islets, suggest comparison with elasmobranchs. In elasmobranchs the ventral ’ The buccohypophyseal canal and its associated rostra1 endocrine tissue in the polypteriformids is not comparable to Latimeria, and recent work (Aler, 1971) has shown that it contains no localization of prolactin cells as would be expected if it were comparable to the canal in elopiform and clupeiform teleosts.

COELACANTH

PITUITARY

143

FIG. 14. Schematic drawings of pituitary glands of Latimeria (A and B) and a hypothetical lower actinopterygian cf Amia (C). A. The vessels of the internal carotids (shaded) surround the elongate rostra1 lobe (RL) and give off direct arterial branches to it (arrows). Aside from a tubular extension of the hypophysial cavity (C), the RL is virtually separated from the remainder of the pars distalis. The RL abuts on the carotid anastomosis distally, while more proximally it is intermittent and represented by small discontinuous islets (IS). The proximal division of the pars distalis is represented by an acidophilic dorsal lobe (DL) and a posterior lobe (PL) of mixed-cell type which has a direct interdigitate contact with the neurohypophysis (shaded). B. This represents a hypothetically compacted Latimeria pituitary, drawn in order to facilitate comparison with lower actinopterygians, C. In forms like Amiu, the presumptive proximal pars distalis maintains a limited NH contact which suggests homology with the PL of Latimeriu. Likewise, the presumptive RPD of Amiu may be The largely gonadotropic ventral portion of the proximal pars distalis in homologous with the DL of Latimeriu. actinopterygian relics and teleosts (PPD-V) may be equivalent to the RL of Lutimeriu. A major difference in pituitary organization between Lutimeriu and the Osteichthyes lies in the presence of a separated, independently vascularized portion of the pars distalis, and in the greater degree of cytologic compartmentalization within the pars distalis (evident in the trilobate organization).

lobe of the pars distalis is characteristically associated with the internal carotid anastomosis in the floor of the chondrocranium, and may retain a tenuous ductlike connection with the more superior portion of the distalis in forms like Heterodontus (Norris, 1941) by a tubular interhypophysial stalk. Like the rostra1

division in Latimeria, the elasmobranch ventral lobe retains a hypophyseal cavity, has a follicular structure and is predominantly basophil in cell type (Della Carte, 1961; Della Corte and Chieffi, 1961). The scant secretory cells noted in the rostra1 division in this immature specimen may suggest that gonadotropic function is localized

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MICHAEL

here, as it is in the elasmobranch ventral lobe. Dr. Olivereau (1974 personal communication) has examined the rostra1 division of an adult ovigerous Latimeria female and noted only large hypertrophic basophils whose cytology was consistent with a gonadotropic function. CONCLUSION

The phylogenetic position of the coelacanth as a crossopterygian might reasonably suggest that pituitary structure in this relic would either resemble that of tetrapods, the descendents of their sister rhipidistians, or be sufficiently generalized so that a tetrapod pattern could be derived from it. However, the coelacanth exhibits two features of pituitary organization which are unknown among the Osteichthyes with which it is classified. These are a tripartite division of the pars distalis, and an associated direct arterial supply to the virtually separated rostra1 division. Relic actinopterygians and lungfishes, in contrast, tend toward a unitary, undivided pars distalis structure with limited segregation of cell types and a single portal vascular supply to the entire pars distalis. The presence in Latimeria of a tripartite pars distalis, and of a ventrally derived and independently vascularized rostra1 division associated with the carotid anastomosis in the chondrocranial floor, are organizational features strongly reminiscent of the elasmobranch ventral lobe, and more remotely the holocephalian Rachendachhypophysis. These remarkable features of pituitary organization together with diverse anatomic characters of a singular nature: the presence of a rectal gland virtually identical to elasmobranchs (Lemire and Lagios, in preparation); the enormous ova and probable ovo-viviparous habitus (Griffith and Thomson, 1973; Millot and Anthony, 1974); the duct-associated pancreatic islet tissue (Grossner, 1968; Epple and Brinn, in preparation); and the

D.

LAGIOS

retention of urea for marine osmolar adaptation (Griffith et al., 1973) are features shared only with the Chondrichthyes - the sharks and their allies -among living fishes. This unique constellation of characters is difficult to reconcile with the conventional interpretation of the coelacanth as allied to crossopterygians and the hypothetical pretetrapod rhipidistian ancestor. Compared with the lungfishes, the coelacanth exhibits numerous differences in basic pituitary structure and organization. These differences do not support a close association between the coelacanth and the Dipnoi in the infraclass Sarcopterygii (the lobe-finned fishes). However, these findings are consistent with the recent systematic reappraisal of the affinities of the Dipnoi and Coelacanthini by Drs. Jarvik (1968) and Bjerring (1973). Based on a reconsideration of the palaeontologic evidence, this interpretation suggests that the lungfishes and coelacanths are unique relic fishes of extraordinary interest to students of comparative disciplines and to those more romantically inclined. Despite its celebration in song and fancy as a kind of missing link, the coelacanth should be viewed as an independent palaeozoic relic incapable of shedding light on the probable structure or biochemistry of the extinct Rhipidistia and their immediate tetrapod descendents. ACKNOWLEDGMENTS The participation of the author in the joint intemational France-Anglo-American Coelacanth expedition to the Comores in February-March, 1972 was made possible by the recommendation of the late Dr. Earl S. Hearld, Steinhart Aquarium, California Academy of Sciences, San Francisco, to whom this work is dedicated. The author sincerely thanks the National Geographic Society and Dr. Edwin Snider, who funded American participation on very short notice; Drs. George and Susan Brown (Department of Fisheries, University of Washington, and chief officers for the Society for the Protection of Old Fishes) who were singularly instrumental in securing the cooperation of the United States Army for my participation; Dr. T. Kerr (Department of Zoology, the University of Leeds) who has encouraged and as-

COELACANTH sisted the author with the expedition and this study and recovered the distal pituitary fragment from the cranium: my comrades-in-arms, Dr. Robert Griffith, Dr. N. Adam Locket, and Dr. Daniel Robineau, particularly Bob and Adam who fixed the tissue for this study; their Excellencies Cheik Said Ibrahim, Prince Nacr-Ed-Dine and Chef du Village Iconi, Mohamed Ali Chabane; and lastly Madi Yousouf Kaar, fisherman of Iconi, Grande Comore.

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