Light microscopy of the pineal organ of two primitive lizards Platyurus platyurus and Hemidactylus frenatus.код для вставкиСкачать
THE ANA’I’OMICAL RECORD 206283-288 (1983) Light Microscopy of the Pineal Organ of Two Primitive Lizards, Platyurus platyurus and Hemidactylus frenatus VERNON L YEAGER, JOHN J TAYLOR, AND PING LUNG CHANG of Medicine, St LOULS, Department ofAnatorny, St Louis IJnzoers~tvSLhool M O 63104‘ A number of structures, such as paraphysis, dorsal sac, parapineal and pineal organ, may develop in the epithalamic region of the brain. While nearly all parts are present in some vertebrates, they may all be rudimentary or lacking in others. Interestingly, the variations do not necessarily follow phylogenic classifications. For instance, the pineal organ is said to be absent in such diverse forms as hagfish (Myxine glutinosa), rays (Torpedo ocellata),crocodiles (Crocodilia),certain owls (Strigiformes). armadillos (Dasypus vellosus), anteaters (Myrmecephagia ,jubota), dolphins (Delphinus longirostris),and whales (Balaenoptera borealis) (Oksche, 1965; Renzoni, 1968). A great diversity in morphology appears among reptiles; where all parts of the complex are said to be absent in crocodiles (Oksche, 19651, only the proximal portion of the pineal organ is present in turtles (Hoffman, 1970; Vivien-Roels, 1969), most parts of the pineal complex are present in the iguana (Tilney and Warren, 19191, and in snakes, the pineal resembles the solid parenchymal organ of certain mammals (Hoffman, 1970; Vivien, 1964). Morphological as well as electrophysiological evidence of a photoreceptive function of the pineal has been provided for fish (Breucker and Horstmann, 1965; Rudeberg, 1971; Dodt, 1963), amphibians (Van de Kamer, 1965; Oksche and von Harnack, 1963; Dodt and Jacobson, 1963), and reptiles (Oksche and Kirschstein, 1968; Steyn, 1960; Miller and Wolbarsht, 1962; Hamasaki and Dodt, 1969).In 1970, Reiter stated, “It is with confidence that scientists now assert that the pineal, in all classes of vertebrates, is capable of modulating endocrine activity. . . .” A light microscopic study of the pineal region of two closely related primitive lizards, Platyurus platyurus and Hemidactylus frenatus, suggests that it is a very simple transitional form and that it may be useful in understanding how the transition from photoreceptor to endocrine function occurred. (c, 1983 ALAN R LISS, INC MATERIALS AND METHODS Two species of gecko, Platyurus platyurus and Hemidactylus frenatus, were identified on the basis of descriptions by Taylor (1963) and used for this study. These lizards were caught in Bangkok, Thailand, and either immediately killed or kept in screen cages in the laboratory. Those which were kept were fed cooked rice, mosquitos, and water ad libitum. The animals varied in age from newly hatched with a snout-vent length of 18 mm to older animals with snout-vent lengths up t o 57 mm. For euthanasia, the lizards were placed in a refrigerator (2-4°C) until a state of hypothermia existed in which no reflexes could be elicted. For some specimens, the head was removed, skinned, and fixed by submersion in buffered neutral 10% formalin. Other specimens were perfused through the heart ventricle with a modified Ringer’s or 0.75% saline solution to which a small amount of heparin was added, followed by 3% paraformaldehyde or 3% glutaraldehyde in phosphate buffer adjusted to pH 7.2. Perfusion was followed by submersion in the same fixative for 2 hours to 2 days. Some of the brains were then removed and placed in wash buffer. After fixation, some whole heads were decalcified in 10% formic acid, embedded in paraffin, and serially sectioned in 8 pm in sagittal or transverse planes while other heads were decalcified in 18% EDTA in 4% buffered (pH 7.2) formaldehyde and embedded in JB-4 embedding mixture (Polysciences, Inc.) for sectioning at 2-3 pm. Tissue sections were stained with luxol fast blue and cresyl violet, hematoxylin and eosin, or periodic acid-Schiff (PAS) reagent. Other epiphyses were removed, postosmicated, dehydrated in alcohol and propylene oxide, embedded in Epon 812, and 0.5 to 1.0-pm Received February 1,1983; accepted March 22, 1983. P.-L.C.’s current address is: Department of Anatomy, College of Medicine, National Taiwan University, Taipei, Taiwan, R.O.C. 284 V.L. YEAGER, J . J . TAYLOR, AND P.-L. CHANG cies studied; therefore, the following decription applies to both. The epiphysis was found to be a small saccular organ attached by connective tissue to the dorsal aspect of the brain between the habenular and posterior commissures (Figs. 1, 2). No communication could be found between the epiphysis and the third ventricle. The epiphysis lies between the posterior extremities of the cerebral hemispheres and the optic tectum. Immediately anterior to the epiphysis were complex foldings of the paraphysis-dorsal sac complex. The latter complex resembled choroid plexus and communicated with the third ventricle by two separate but closely related ducts. A large venous plexus was closely related to the epiphysis. The epiphysis had a thin connective tissue capsule which was continuous with the pia mater of the epithalamus. The capsule consisted of collagen and reticular fibers with numerous fibroblasts and a few mast cells and pigment cells. The outer surface of the capsule had a more regular outline than its inner surface, which had occasional septa projecting into small infoldings of the epithelial lining of the epiphysis. Sometimes the outer surface exhibited a n indentation, suggesting a multivesicular structure. There was no indication of a connective tissue or fibrous stroma separating epiphyseal cells. A few myelinated nerve fibers were seen in the capsule, but these were rare. No ganglion cells were seen in the capsule or any other location in the epiphysis. Immediately inside the capsule were numerous small blood vessels, most of which appeared to be capillaries (Figs. 3, 4). The epithelial cells were separated from the capillaries by a very narrow PAS-positive basement membrane. When there were infoldings of the epithelium, the connective tissue septa contained blood vessels also. The epithelial lining gave the appearance of a pseudostratified columnar epithelium, but since the outer limiting cell membranes were not stained by any of the techniques used, this could not be established. In most Fig. 1. Sagittal section of brain showing cerebral hemisphere (CH), paraphysis (P), dorsal sac (DS), epi- physis (Et, optic tectum (OT), and cerebellum (C). Paraf fin section, luxol fast blue stain. X 42. sections were cut on a n ultramicrotome and stained with toluidine blue. RESULTS No differences were found in the two spe- LIZARD PINEAL ORGAN 285 Fig. 2. Section of an epiphysis showing its vesicular structure. The capsule (Ca), numerous blood vessels (arrows), epithelium, and debris in the lumen (L)are clearly seen. Portions of the paraphysis-dorsal sac complex (PDS) are included. Epon section, toluidine blue stain. epon sections studied with the light microscope, the epithelium was seen to differ in its outer, intermediate, and inner zones (Fig. 3). The outer or basal zone was made up primarily of cytoplasm and intercellular spaces. In some perfused specimens, these spaces were enlarged and extended into the other zones. Specimens fixed by immersion had spaces the size of capillary lumina or smaller. None of the spaces had distinguishable linings. Very few nuclei were found in this zone. The blood vessels were located at the base of this zone so that three sides of the capillaries were related to the epithelium and one side to the connective tissue capsule (Fig. 4). The intermediate zone was about twice the thickness of the outer or basal zone and consisted mostly of closely packed nuclei. These nuclei were round, oval or tear-drop shaped. Typically there were prominent nucleoli within the pale nucleoplasm. Chromatin was found adjacent t o the nuclear membrane. Intercellular spaces were not evident in this layer in tissues fixed by immersion. The cytoplasm was scanty in this zone and not remarkable. Occasional very dense bodies were noted in the supranuclear cytoplasm of this zone, but these bodies were more characteristic of the inner zone. Most of the nuclei and cells of this zone appeared t o be similar, but occasional very dense cells were noted (Fig. 5 ) . In some of these cells, only the nucleus was more intensely stained, but in others, both the cytoplasm and nucleus were stained more deeply than the typical cell. The inner zone adjacent to the lumen was the thinnest zone. It had nuclei, but not in the numbers seen in the intermediate zone. X 215. Fig. 3. Higher magnification of the wall of the epiphysis from a specimen fixed by immersion. Note t h e capsule (Ca), blood vessels (arrows), intercellular spaces (I) in the outer zone, packed tear-shape6 nuclei in the Most of the nuclei were somewhat smaller than those of the intermediate zone and the chromatin was more clumped. Many of these had nuclear bodies or inclusions (Fig. 4).In some areas of some of the epon sections, a series of stained dots appeared parallel to the lumen in the apical portions of the cells (Fig. 3). In obliquely cut epithelia, a lattice pattern indicated that these probably represented terminal bars or cell junctions. The very dense bodies described in the intermediate zone were especially common in this zone. In some specimens, there appeared to be one of these bodies in every cell. The bodies were stained by toluidine blue but not by hematoxylin or PAS treatment. No detail could be seen within the bodies even when lightly stained sections were studied. The luminal surface of the epithelial cells was rounded or irregular, and material in the lumen seemed to indicate that degenerating cells, cell parts, or secretory material was being extruded into the lumen. A slight PAS reaction occurred a t the lumen surface or immediately deep to it. The lumen of the epiphysis appeared to be an actual fluid-filled space rather than a potential or artifactual space. DISCUSSION If the region between the habenular and posterior commissures gives rise to only one intermediate zone, and the inner zone adjacent to the lumen (L). The series of small dots in the inner zone probably represent junctional complexes. Epon section, toluidine blue stain. x 750. vesicular structure, as is the case for the two species of gecko studied in this report, some problem in identification may occur, since end-vesicles and proximal portions for both parapineal organs and pineal organs have been described for lizards. Tilney and Warren (1919) state that for Gecko versus and Platydactylus muralis only the epiphysis is present, whereas for Hemidactylus uerruculatus they mention only a n end-vesicle, and for Hemidactylus mabouia and Gehyra oceanica they mention a n end-vesicle and a stalk. It is not clear whether they studied the pineal region of geckos themselves or simply reviewed the findings of others. Since the structure described in this report resembles in position and shape the proximal portion of the complex in other forms where the endvesicle is also present, and because the proximal portion is the most constant part of the pineal complex, the present authors believe it is the proximal portion of the pineal organ and the homolog of the mammalian epiphysis cerebri. Additional studies, especially Fig. 5. Specimen showing dense cells. The three zones are indicated by lines (- - -) near the right side of the figure. Note also t h e difference in appearance of many of the nuclei in the inner zone compared to those of the intermediate zone. Epon section, toluidine blue stain. x 980. Fig. 4. Higher magnification from a specimen fixed by perfusion. Note the dilated blood vessels IBV), intercellular spaces (I), supranuclear dense bodies (DB), nu- cleus with inclusion body (arrow), and clublike cell processes projecting into t h e lumen (L). Epon section, toluidine blue stain. x 1830. 5 288 V.L. YEAGER, J.J.TAYLOR, AND P.-L. CHANG of embryological specimens, are needed to definitely prove its identity. To question whether the epiphysis of these geckos is functioning as a photoreceptor organ, a n endocrine organ, or both cannot be answered by a light microscopic study. The cell shape and arrangement is similar to that found in lower forms in which photoreception still occurs, but outer segments could not be identified. In thin plastic sections of the retina of the lateral eye of these lizards, the outer segments were very apparent, but nothing comparable was seen in the pineal. Ganglion cells were never identified, nor was a pineal nerve found, although two or three myelinated fibers were seen in or near the capsule in a few specimens. Pigment cells, which form a prominent feature of the parietal organ of some reptiles (Tilney and Warren, 1919; Oksche and Kirschstein, 19681, occurred in very small numbers. Therefore, morphologic evidence for photoreception was not convincing. In a number of saccular epiphyses the lumen communicates with the third ventricle (Ralph, 1970; Hafeez, 1971; Rudeberg, 1971). In this study, both the paraphysis and the dorsal sac were found to have long attenuated ductlike communications with the third ventricle, but careful examination of 2pm thick serial sections revealed no communication between the ventricular system and the epiphysis. If the fluid-filled cavity of the epiphysis receives secretory material, there does not appear to be a ready exit for it. A possible route for secretory products, if they exist, would seem to be the intercellular spaces in the basal zone adjacent to the capillaries. Quay (1974) has shown by India ink perfusions that intercellular canaliculi exist in the rat pineal and he suggests that they constitute a transport route between pinealocytes and blood vessels. The number and size of the capillaries suggest that the cells of the epiphysis in the gecko pineal are metabolically active. Whether the gecko epiphysis cerebri functions as either a photoreceptor or an endocrine organ could not be established in this light microscopic study. ACKNOWLEDGMENTS The assistance of Mrs. Amara Kumnirdsena Megpaibul and Mrs. Wantanee Trakulrungsi is gratefully acknowledged. LITERATURE CITED Breucker, H., and E. Horstmann (1965) Elektronenmikroskopische Untersuchungen a m Pinealorgan der Regenbogenforelle ~Salrno irideus). Prog. Brain Res., 20959-269. Dodt, E. (1963) Photosensitivity of the pineal organ in the teleost, Salrno irideus (Gibbonsl. Experientia, 19,642-643. Dodt, E., and M. Jacobson (1963) Photosensitivity of a localized region of t h e frog diencephalon. J. Neurophysiol., 265’52-758. Hafeez, M.A. (1971) Light microscopic studies on the pineal organ in teleost fishes with special regard to its function. J. Morphol., 134r281-314. Hamasaki, D.I., and E. Dodt (1969) Light sensitivity of the lizard’s epiphysis cerebri. Pflugers Arch., 313:1929. Hoffman, R.A. (1970)The epiphyseal complex i n fish and reptiles. Am. Zool., lOt191-199. Miller, W.H., and M.L. Wolbarsht (1962) Neural activity in the parietal eye of a lizard. Science, 135,316-317. Oksche, .A. (1965)”Survey of t h e development and comparative morphology of the pineal organ. Prog. Brain Res., 10:3-29. Oksche, A,, and H. Kirschstein (1968) Unterschiedlicher elektronenmikroskopischer Feinbau der Sinneszellen im Parietalauge und im Pinealorgan (Epiphysis cerebri) dcr Lacertilia. Ein Beitrag zum Epiphysenproblem. Z. Zellforsch., H7r159-192. Oksche, A,, and M. von Harnack (1963) Elektronenmikroskopische Untersuchungen am Stirnorgan von Anuren. 2 . Zellforsch., 59:239-288. Quay, W.B. (1974) Pineal canaliculi: Demonstration, twenty-four-hour rhythmicity and experimental modification. Am. J. Anat., 139:81-94. Ralph, C.L. (1970)Structure and alleged function of avian pineals. Am. Zool., I0:217-235. Keiter, K.J. (1970) A precis of the pineal odyssey. Am. Zool., 10t189-190. Renzoni, A. (19681 Osservazioni comparative sull’epifisi degli Strigiformi ed Ordini affini. Arch. Ital. Anat. Ernbriol., 73,321-336. [Cited from Ralph, C.L. (1970) Am. Zool., 20:217-2:35.) Rudeberg. C. (1971) Structure of the pineal organs of A nguilln anguilla L. and Lebifes reticulatus Peters (Teleosteii. Z. Zellforsch., 222r227-243. 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