Estudou-se com a microscopia eletrônica os efeitos do autotransplante da parte intermédia da pituitária do cadoz de água salgada, Gullichthys mirabilis. No peixe sem transplante, a parte intermédia possui dois tipos de células secretoras: hematoxilina de Pb-positiva (PbH+) e hametoxilina de chumbo negativa (PbH-), diretamente inervadas pelo axônios neurossecretores (tipo A e B) originários do hipotálamo

Bern, Howard A.

Nagahama, Yoshitaka

Nishioka, Richard S.

English

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Bol. Zool. e Biol. Mar., N .S . ,  n.° 30, pp. 69-79, São Paulo, 1973PRELIMINARY REPORT ON THE EFFECT OF PITUITARY AUTOTRANSPLANTATION ON THE PARS INTERMEDIA OF THE SEAWATER GOBY,G1LLICHTHYS MIRABILIS1RICHARD S. NISHIOKA, YOSHITAKA NAGAHAMA and HOWARD A. BERNDepartment of Zoology and its Cancer Research Labo­ratory, University of California, Berkeley, California 94720.RELATO PRELIMINAR SOBRE OS EFEITOS DO AUTOTRANSPLANTE DA PITUITÂRIA NA PARTE INTERMÉDIA DO CADOZ DE ÂGUA SALGADA,GILLICHTHYS M IRABILISRESUMOEstudou-se com a microscopía eletrônica os efeitos do autotrans- plante da parte intermédia da pituitaria do cadoz de água salgada, GiUichthys mirabilis.No peixe sem transplante, a parte intermédia possui dois tipos de células secretoras: hem atoxilina de Pb-positiva (PbH +) e hema- toxilina de chumbo negativa (P b H -), diretam ente inervadas pelos axónios neurossecretores (tipo A e B) originarios do hipotálamo.Após o autotransplante, a parte intermédia revasculariza-se ra­pidamente e urna semana após o transplante as células PbH+ pos­suem pequeno número de grânulos secretores, retículo endoplasmá- tico tortuoso e bem desenvolvido.Depois de quatro semanas as células PbH+ aparecem bem ativas e após quatro m eses m uitas vesículas secretoras acumularam-se ñas células, embora algumas ainda possuam muito retículo endoplasmá- tico tortuoso, como nos peixes controles.1 W ith this paper w e express our respect and affection for Professor Paulo  Sawaya and our appreciation of his contributions to the field of comparative physiology. The research was aided by N ational Science Foundation Grant GB 35239X and National Institute of Health Grant CA-05388.As células P b H - respondem de forma similar. É possível que o controle hipotalâmico sobre a parte intermédia é restabelecido v ia  circulação sistêmica.ABSTRACTThe effect of autotransplantation of the pars interm edia of the pituitary of the seawater goby, G illichthys m irabilis, was studied by electron microscopy. In intact fish, the pars intermedia contains two types of secretory cells: lead hem atoxylin-positive (PbH +) and lead hem atoxylin-negative (P b H -) cells, directly innervated by neuro­secretory axons (types A and B) from the hypothalamus. After autotransplantation, the pars intermedia was revascularized rapidly. One week after transplantation, the PbH+ cells contained small numbers of secretory granules and well-developed rough endoplasmic reticulum. After four weeks, the PbH+ cells appeared very active; after four months many secretory vesicles had accumulated within  the cells, although some cells still contained much rough endo­plasmic reticulum, as in intact fish. The P b H - cells responded similarly. It is possible that hypothalamic control over the pars intermedia is reestablished via the systemic circulation.INTRODUCTIONThe pars intermedia of the teleost pituitary is composed of two distinct secretory cell types: one stains with lead hematoxylin (PbH+ cells) and the other does not stain with this dye but does react with the periodic acid Schiff (PAS) method and are referred to herein as PbH- cells (cf. Ball and Baker, 1969; Sage and Bern, 1972). The PbH+ cells appear to secrete MSH, whereas the PbH- cells have an uncertain function, possibly related to osmoregulation (Olivereau and Ball, 1970). These two cell types are directly innervated by two kinds of neurosecretory fibers: types “A” and “B” The nature of the regulation accomplished by these axons is of obvious interest. Recently, some morphological studies have begun to elucidate this problem (Zam­brano et al., 1972; Follenius, 1972), and the hypothalamic control of the pars intermedia in teleost fishes has been recently reviewed (Ball et a l 1972).Some light-microscope studies have been carried out on the pars intermedia transplanted away from the hypothalamus (Poecilia for­mosa by Ball et a l 1965, and by Olivereau and Ball, 1966; Poecilia latipinna and Anguilla anguilla by Olivereau, 1969; Gasterosteus aculea- tus by Leatherland, 1970). However, the information accumulated todate is insufficient to provide a complete understanding of hypothala­mic control of the teleost pars intermedia. The effect of transplanta­tion on the rostral pars distalis of the Gillichthys pituitary has been reported (Nagahama et al., 1973). Incidental to that study, we decided to examine the effects of autotransplantation on the cells of the pars intermedia.MATERIALS AND METHODSGillichthys mirabilis, a gobiid teleost species from San Francisco Bay, were held in seawater tanks at 14°C and fed on live brine shrimp. All fish were acclimated for a minimum of one week before auto­transplantation. The intact pituitary from anesthetized fish was care­fully removed and transplanted into a prepared cavity under the tongue (Nagahama et al., 1973). In this position the grafted pituitary is clearly visible through the oral mucosa and the course of revascula­rization, which takes about a week, can be followed.A total of 7 controls and 14 autotransplanted fishes were examined by electron microscopy. Eight fishes were killed after 1-2 weeks, four fishes after 4 weeks and two fishes after 4 months of auto-transplanta­tion. The pituitary was removed and initially placed in a modified glutaraldehyde,/paraformaldehyde fixative. Cacodylate-buffered osmium tetroxide was used for post-fixation, and the tissues were dehydrated in graded ethanols and embedded in epoxy resin (Spurr, 1969). Thick sections (1 ¡x) were cut and stained with methylene blue-azure II (Ri­chardson, et al., 1961). Thin sections were cut with glass and diamond knives and studied in a Siemens Elmskop II or Phillips 200 electron microscope.RESULTSIntact fish. The intact pituitary is located within a depression in the tuberal region of the hypothalamus of Gillichthys. Neurohypophys­ial tissue stainable with aldehyde fuchsin covers the outer surface of the posterior part of the pituitary which comprises the pars intermedia (Fig. 1). Neurohypophysial processes also penetrate among the cells of the pars intermedia. Of the two cell types in the pars intermedia, the lead hematoxylin-positive type (PbH+) is more numerous. The other cell type does not stain with lead hematoxylin (PbH~).In 1 /¿-thick epoxy sections stained with methylene blue- azure, the overall distribution of the pars intermedia cells as well as the ramifications of the neurohypophysial tissue could be clearly observ­ed (Fig. 1).U ltrastructurally, numerous axons filled with type A granules are found a t the periphery of the pars intermedia. Type A axons are also found among the pars intermedia cells, and some axons are indented deeply into the cells. In addition, type B axons, less numerous and smaller in size, are also found among the pars intermedia cells (Fig. 4).The major cell type (PbH+) contain large pale granular vesicles, filling the cytoplasm of most of the cells along with some rough endo­plasmic reticulum (Fig. 4). A small proportion of these cells in control fish exhibit multilayered endoplasmic reticulum.The second cell type (PbH“ ) contain dense spherical granules which fill most of the cytoplasm (Fig. 4). Based on ultrastructural criteria, these cells show low to moderate activity. Their processes are narrow and tenuous, superficially resembling type A axons.One to two weeks after autotransplantationOne to two weeks after autotransplantation, the neurohypophysial tissue shows variable signs of disintegration (Fig. 2). Most of the axons appear within vacuoles of the phagocytes which apparently invade the transplanted pituitary (Fig. 5).Most PbH+ cells show extensive development of rough endoplasmic reticulum, which takes the form of concentric whorls as well as other multilayered arrangements (Fig. 5). These highly active-appearing cells contain relatively few secretory vesicles. These is an indication of greater development of the endoplasmic reticulum in the vicinity of massive neurohypophysial disintegration. However, a few PbH+ cells contain many secretion granules essentially similar to those found in control pituitaries.The PbH- cells appeared more active and contained more endo­plasmic reticulum than in the control pituitaries, and they contained fewer dense granules (Fig. 5).Fig. 1  Cross section of pars intermedia of seawater Gillichthys. Neurohypophysisforms distinctive layer at periphery. Secretory cells are pale and cellular borders are indistinct. 1 y methylene blue-azure. 400 x .Fig. 2 __ Cross section of pars intermedia of 2 w eek autotransplanted seawaterfish. N ote absence of neurohypophysial tissue and invasion of phagocytes (Ph)among darker staining secretory cells. 1 y methylene blue-azure. 400 x .Fig. 3 __  Cross-section of pars intermedia of 4 month autotransplanted seawaterfish. N ote presence of phagocytes (Ph) in connective tissue at periphery but absence from interior of pars intermedia. Secretory cells appear pale as incontrol (Fig. 1). 1 y m ethylene blue-azure. 400 x .Fig. 4 — Electron micrograph of seawater G illichthys pars intermedia. Portions of P b H - and PbH+ cells are shown. Note types A and B neurosecretory axons from neurohypophysis (NH) .  20,000 x .Fig. 5 __ Electron micrograph of seawater Gillichthys pars intermedia 2 weeksafter autotransplantation. Note extensive endoplasmic reticulum  in PbH+ cell. Phagocyte (Ph) contains neurohypophysial debris. P b H - cell contains dense granules and some endoplasmic reticulum. 8,000 x .Four weeks after autotransplantationThe pars intermedia of pituitaries transplanted for four weeks m aintains a high degree of cellular activity as indicated by the quantity of rough endoplasmic reticulum. The size of some of the concentric whorls is somewhat diminished, but there appear to be more of them. Only a few phagocytes occur among the granular cells, although many are still present a t the periphery of the transplant. A few of the PbH+ cells are very osmiophilic and appear to be undergoing degeneration. The PbH- cells also show undiminished cellular activity.f ig .  6 — Electron micrograph of seawater G illichthys pars intermedia 4 months after autotransplantation. Note dilated secretory vesicles in PbH+ cell near capillary (Cap). Small P b H - cell and process are present. 8,000x .Four months after autotransplantationFour months after transplantation, very few phagocytes are seen within the pars intermedia, although many are still present around the capsule (Fig. 3). Many PbH+ cells are large and full of secretory vesicles and appear superficially similar to control cells. On closer examination, however, these secretory vesicles are larger and contain less granular material (Fig. 6). Other cells also show an extensive endoplasmic reticulum. A few large osmiophilic inclusions are evident in some PbH+ cells. In 1 ju, sections many of the secretory cells appeared larger than in the controls. Most of the PbH~ cells contain many large dense secretion granules. When present, the rough endoplasmic reti­culum forms layers around the nucleus. These cells possess tortuous processes which occupied some of the interstitial space between PbH+ cells (Fig. 6). Some smaller cells contain fewer granules, but otherwise appear active.DISCUSSIONThe pars intermedia in Gillichthys mirabilis as in other teleosts contains two cell types, one of which is lead-hematoxylin-positive (PbH+) and the other is either periodic acid Schiff-positive or lead hematoxylin-negative (PbH~), depending on the species (cf. Ball and Baker, 1969; Sage and Bern, 1972). Electron microscope studies show that types A and B axons make direct contact with both cell types. In intact fish both cell types are presumably controlled to a major degree by these axons. When the physical connection with the hypo­thalamus is lost following pituitary transplantation, both axonal types completely disintegrate within two weeks, and the transplanted pitui­tary  cells are consequently without direct hypothalamic control.The notable hypertrophy of the rough endoplasmic reticulum of PbH+ cells one to two weeks after autotransplantation suggests lifting of inhibition, ascribable to the disintegration of the aminergic type B fibers; similar changes occur after 6-hydroxydopamine treatm ent which selectively destroys type B fibers (Zambrano et al., 1972). The effect of type A axon degeneration is unknown. In contrast to Gilli­chthys, in Poecilia formosa homotransplants, the PbH+ cells became atrophied and scarce (Olivereau and Ball, 1966). One to two weeks after homotransplantation in Gasterosteus aculeatus, the grafted chro­mophobes (possibly equivalent to our PbH+ cells) became significantlysmaller than normal (Leatherland, 1970). On the other hand, in P. latipinna, autotransplants are well maintained a t least for 3 weeks and show moderate activity (Olivereau, 1969). Thus, the response of the PbH + cells after pituitary transplantation appears to vary with the species studied.Many PbH + cells in the transplants showed a considerable number of secretory vesicles after four months, resembling to some degree the condition which exists in intact fish. Maintenance of PbH+ cells in autotransplants after two months has also been reported in Anguilla (Olivereau, 1969). The possibility of distant control of the pars inter­media by the hypothalamus cannot be discounted, although direct evidence is lacking as yet. Inasmuch as the axonal processes of the preoptic neurons (type A fibers) and the lateral tuberal neurons (type B fibers) were transected during hypophysectomy prior to transplan­tation, formation of a neurohemal structure by the proximal axon stumps is highly possible (Sathyanesan, 1965). Control of the ectopic pituitary could then be achieved via the systemic circulation.There are relatively few PbH- (electron-dense granule-containing) cells in intact Gillichthys. These cells were also found four months after autotransplantation. Crude estimates based on 1 ¡± sections and on electron micrographs indicate tha t there was no decrease in the number of these cells. Therefore, it is possible tha t neither stimulatory nor inhibitory factors directly control the functional activity of these cells. Our observations differ from those on Poecilia formosa (Olive­reau and Ball, 1966), P. latipinna (Oliveireau, 1969) and Gasterosteus (Leatherland, 1970) in tha t the Gillichthys cells remain active.This preliminary study opens avenues for further investigation. The possibility of systemic control of the autotransplant by the hypo­thalamus via a regenerated neurohemal structure is raised. Functional significance of this new control system can be examined by challenging autotransplanted fish with light and dark backgrounds in order to see how rapidly the pars intermedia cells are able to respond. Further experiments are now in progress.R E F E R E N C E SBALL, J. N. and BAKER, B. I. (1969) — The pituitary gland: anatomy and histophysiology. In  “Fish Physiology” (W. S. Hoar and D. J. Randall, eds.), Vol. II, pp. 1-110. Academic Press, N ew  York and London.BALL, J. N.; OLIVEREAU, M.; SLICKER. A. M. and KALLMAN, K. D. (1965) — Functional capacity of ectopic pituitary transplants in the teleost Poecilia  formosa, w ith a comparative discussion on the transplanted pituitary. Phil. Trans. B 249:69-99.BALL, J. N.; BAKER, B. I.; OLIVEREAU, M. and PETER, R. E. (1972) — Investigations on hypothalamic control of adenohypophysial functions in teleost fishes. Gen. Comp. Endocrinol., Suppl. 3:11-21.FOLLENIUS, E. (1972) — Ultrastructure of the hypophysial aminergic nerves in the teleost G asterosteus aculeatus degenerating after application of 6-hydroxydo- pamine. Z. Zellforsch., 128:69-82.LEATHERLAND, J. F. (1970) — Histological investigation of pituitary homo­transplants in the marine form (trachurus) of the threespine stickleback, G asterosteus aculeatus L. Z. Zellforsch. 104:337-344.NAGAHAMA, Y.; NISHIOKA, R. S. and BERN, H. A. (1973) — Structure and function of the transplanted pituitary in the seawater goby, G illichthys viira- bilis. I. The rostral pars distalis. (Submitted for publication).OLIVEREAU, M. (1S69) — Activité de la pars intermedia de l ’hypophyse auto- transplantie chez l ’Anguille. Z. Zellforsch. 98:74-87.OLIVEREAU, M. and BALL, J. N. (1966) — Histological study of functional ectopic pituitary transplants in a teleost fish (Poecilia form osa). Proc. Roy. Soc. B 164:106-129.OLIVEREAU, M. and BALL, J. N. (1970) — Pituitary influences on osmoregu­lation in teleosts. Mem. Soc. Endocrinol. 18:57-85.RICHARDSON, K. C.; JARETT, L. and FINKE, E. H. (1961) — Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 35:313-323.SAGE, M. and BERN, H. A. (1972) — Cytophysiology of the teleost pituitary. International Review of Cytology 31:339-376.SATHYANESAN, A. G. (1965) — The reorganization of the hypophysial stalk  follow ing hypophysectomy in the teleost Porichthys notatus Girard. Z. Zell­forsch. 67:734-739.SPURR, A. R. (1969) — A low-viscosity epoxy resin embedding medium for electron  microscopy. J. Ultrastruct. Res. 26:31-43.ZAMBRANO, D.; NISHIOKA, R. S. and BERN, H. A. (1972) — The innervation  of the pituitary gland of teleost fishes. In “Brain-Endocrine Interaction. Median Eminence: Structure and Function. Int. Symp. Munich 1971”, 50-66. Karger, Basel.

Preliminary report on the effect of pituitary autotransplantation on the pars intermedia of the seawater goby, Gillichthys mirabilis

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Preliminary report on the effect of pituitary autotransplantation on the pars intermedia of the seawater goby, Gillichthys mirabilis

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