THE AMERICAN JOURNAL OF ANATOMY 179:211-219 (1987) Opossum Adrenal Medulla: I. Postnatal Development and Normal Anatomy STEPHEN W. CARMICHAEL, DANIEL B. SPAGNOLI, RICHARD G. FREDERICKSON, WILLIAM J. KRAUSE, AND JAMES L. CULBERSON Department o f Anatomy, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905 (S.W. C.); Department o f Anatomy, West Virginia Uniuersity, Morgantown, West Virginia 26506 (D.B.S., R. G.F, J.L. C.); Department ofdnatomy, University of Missouri, Columbiq Missouri 65212 (IKJ.K.) ABSTRACT The anatomy and histology of the adrenal gland in the adult opossum were found to be typical for mammals. The development of the adrenal medulla was also found to follow the typical mammalian pattern. Primitive sympathetic cells were found in both intra- and extra-adrenal locations in the newborn at a time when chromaffin precursor cells were migrating to the adrenal anlage. Pheochromoblasts first appeared within the forming medulla where at a later stage chromaffin cells could be observed forming columns of cells between adjacent sinusoids. Unlike in other mammals, much of this development takes place postnatally when the neonate is in the mother’s marsupium. The value of the developing opossum adrenal medulla as an experimental model is stressed, since a significant amount of development takes place in an environment that is accessible to experimental manipulation. INTRODUCTION It is well known that catecholamine-containing cells are widely distributed throughout the nervous system, including the central and peripheral portions (Cooper et al., 1982). As pointed out by Black (19821, the peripheral autonomic nervous system has provided a relatively simple approach to the study of cellular diversification and specialization within an organism. In turn, the adrenal medulla provides a rather homogeneous collection of catecholamine-containing cells that are relatively accessible and have proven to be a valuable model for biological studies (Carmichael and Winkler, 1985). The embryonic origin of the adrenal medulla and related cells has been widely studied as a model of how neurotransmitters are formed and sequestered within their cellular environment (for reviews see Andrew, 1981; Coupland, 1980). Most of the experimental developmental studies on the adrenal medulla have been done on avians or amphibians since the tissue is readily accessible. While there have been several descriptions of development in mammals, such as rabbit (Coupland and Weakley, 19681, rat (Elfvin, 1967; El-Maghraby and Lever, 1980), and man (Hervonen, 1971),there have been no experimental studies on the developing mammalian organ. It was decided to examine the development of the adrenal medulla in marsupials, because these species may provide accessible experimental models for developmental stud@ 1987 ALAN R. LISS, INC. ies (Moore and Bodian, 1940; Krupp and Quillin, 1964; Jurgelski, 1974). The North American opossum (Didelphis uirginiana) is the only marsupial that is native to North America. It is a generalized metatherian mammal that has remained essentially unchanged since its evolution during the Cretaceous period (Colbert, 1967). It has a unique gestational period that includes 13 days (k 6 hours) of intrauterine development, and at birth it is a mixture of embryonic and fetal tissues (Jurgelski et al., 1976). During the 60- to 70-day period following birth, the immature pouch-young undergo late embryonic and fetal development in the mother’s marsupium. This has been shown for a number of organ systems, including endocrine organs such as the pituitary (Sherman and Dunkerley, 1982) and thyroid (Krause and Cutts, 1983).Thus, the neonatal opossum is accessible and potentially can be used in controlled, quantitative, experimental investigations of development. This study was undertaken to describe the normal postnatal development of the adrenal medulla in the opossum and the normal anatomy of this organ. We were particularly interested in determining to what extent the adrenal medulla develops postnatally. The biogenesis of the chromaffin vesicle and related ultrastructural aspects will be described in the paper to follow. MATERIALS AND METHODS Adult female opossums (Didelphus uirginiana) were captured in the wild (near Morgantown, WV, or Columbia, MO) and caged for 2 weeks. They were checked daily for pouch-young. Neonatal opossums ranging from newborn to 12 days old were examined. In addition, some adult opossums were studied for the normal anatomy. Adults were anesthetized with 50 mgkg of nembutal (IP) and newborns were anesthetized with hypothermia by placing them on ice. Newborns were perfused with fixative through the left ventricle by following a published procedure (Spagnoli et al., 1979). Other neonates were fixed by immersion in 3% glutaraldehyde with phosphate buffer (pH 7.4). Animals that were prepared for light microscopic examination were perfused with a solution containing 2.5% potassium dichromate and 10% formalin at pH 4.1 (Wood, 1963). Some specimens for light microscopy were fixed with 3% glutaraldehyde, postfixed with osmium tetroxide, embedded in EponAraldite, sectioned at 1 pm, and stained with 1%tolu- Received July 5, 1985. Accepted April 10, 1986. 212 S.W. CARMICHAEL ET AL. Fig. 1. Section of the adrenal gland of an adult opossum. This figure shows the basic organization of the adult adrenal. G, zonaglomerulosa; F, zona fasciculata; R, zona reticularis; M, medulla. H and E. X 117. Figs. 2,3.The medullary and cortical regions of the adrenal glands of adult opossums are histochemically delineated. Fig. 2. A section through an adrenal gland fixed with potassium dichromate. Cells in the medullary region (M) are darkly impregnated by the chromaffln reaction, whereas the cortical cells (C) are nonreactive. Unstained. X 70. Fig. 3. Cells in the medullary region (M) are intensely fluorescent, whereas those in the cortex (C) are nonfluorescent. Fluorescence: glyoxylic acid-induced. X117. idine blue. In addition, some specimens were examined for fluorescence by using the sucroseipotassium phosphatelglyoxylic acid technique of de la Torre and Surgeon (19761, and others were subjected to a n argentaffin reaction using ammoniacal silver (F'rederickson et al., 1978). Neonates to be examined by scanning electron microscopy were fixed by immersion; the posterior abdominal wall was exposed; then the specimens were dehydrated in alcohol and critical-point dried with COz. Additional specimens were prepared for transmission electron microscopy and are described in the paper to follow. cord of chromaffin cells passes through the cortex in association with a n arteriole. Chromaffin cells within the cord are in direct contact with cortical cells (Fig. 2). RESULTS Normal Anatomy of the Adult Adrenal Fig. 4. The relationships of the right and left adrenal glands (A) of the newborn opossum to the root of the mesenetry (M), metanephros (MT), mesonephros (ME), inferior vena cava (IVC), and liver (L). The adrenal glands are larger than the metanephri. X23. Fig. 5. The adrenal gland and associated structures: A, adrenal gland; EA, extra-adrenal chromafin tissue; M, mesentery; arrow, inferior vena cava; MT, metanephric tubules. Toluidine blue. x 152. Fig. 6. Serial section that follows Figure 5. The majority of chromaffin tissue is pale staining and slightly granular (CR).A few chromaffin cells located near sinusoids have dark cytoplasmic argentaffin granules (arrows extending from CR). C, Cortical tissue; (N, unmyelineated nerves; arrow; inferior vena cava; and S, sinusoids. Ammoniacal silver. The right adrenal gland is medial and rostra1 to the upper pole of the kidney. It is directly adjacent to the dorsal aspect of the inferior vena cava and the inferior I\1y,_ Y'x7 aspect ofthe right lobe of the liver. The left adrenal is medial to the upper pole of the left kidney. The adrenal Figs. 7,8.Extra-adrenal chromafin tissue is shown in Figure 7; of the opossum are encapsulated within connec- chromafin tissue that has invaded the adrenal gland is shown in Figure 8. Primitive sympathetic cells (PSC) or cells with dark basotive tissue that at the hilus contains nerve fibers, gan- philic nuclei and a thin band of cytoplasm, pheochromoblasts (PI, and glion cells, and large veins. Numerous arterioles are mitotically active precursors (MI are present. Mitotically active cells also located within the capsule, and some can be fol- are most frequently observed in the extra-adrenal chromafin tissue. lowed into and through the cortex to the medulla. Three and E. x585. cortical zones are present: a compact glomerulosa, a Fig. 9. Micrographs of a 10-pm cryostat section prepared to demonzona fasciculata with long, straight cords, and a zona strate catecholamine histofluorescence. Orientation can be achieved by~comparing 5 and 9. Extra- and intra-adrenal (A)regions are reticulariswith anastornotic cords @ig. 1).~ ~ d ~ t hroughly l separated i ~ Figures ~ by - the white line. Chromafin tissue is fluorescent, lined sinusoids from the subcapsular region pass be- whereas intermingled cortical tissue and stromal structures are nontween the cortical cords to the medulla. Infrequently a fluorescent. Fluorescence: glyoxylic acid-induced. x 117. POSTNATAL DEVELOPMENT OF OPOSSUM ADRENAL MEDULLA 213 214 S.W. CARMICHAEL ET AL Fig. 10. Although the adrenal glands (A) in 8-day-old opossums are much larger than those in the newborn, they are relatively small when compared to the metanephri @IT). ~ 2 3 . Fig. 11. A number of chromaffn cells with dark cytoplasmic argen- t a f i n granules (arrows from CR) are observed in extra-adrenal (EA) and intra-adrenal locations. Other chromaffin tissue cells, such as those in the vicinity of CR, are only slightly granular and pale staining. C, cortex. Ammoniacal silver. X215. The parenchyma of the adrenal medulla is formed by anastomotic cords of chromaffin cells. The medulla is surrounded by cortex, except at the hilus where medullary cords are associated with the connective tissue of the capsule. The stroma of the medulla consists of connective tissue associated with nerves, arterioles, or capillaries, and endothelium-lined venous sinusoids. In the tissue that was fixed with glutaraldehyde and osmium and embedded in plastic, two populations of chromaffin cells could be identified. When stained with toluidine blue, one population had dark-blue to bluegreen cytoplasmic granules, whereas the other population had pale blue granules. Nuclear characteristics, including a prominent nucleolus and clumped chromatin, were common to both cell types. Glyoxylic-acid-induced catecholamine fluorescence was localized within the cytoplasm of chromaffin cells (Fig. 3). Histologically, precursor chromaffin cells can be seen both within the developing adrenal anlagen and in locations between the dorsomedial borders of the anlagen and the aorta and vena cava (Fig. 5). Intermingled cortical and precursor chromaffin cells at various stages of development form the parenchyma of the adrenal at this stage (Fig. 6). Two cell types can be identified in extraadrenal (Fig. 7) and intra-adrenal (Fig. 8) chromaffn tissue, where the presence of catecholamines is demonstrated by fluorescence microscopy (Fig. 9). The predominant cell during the first 3 days is the primitive sympathetic cell (PSC), which has an intensely basophilic nucleus and a thin rim of cytoplasm. The other cell type, which has a less basophilic nucleus, noticeably more cytoplasm, and variable cytoplasmic granularity, is a pheochromoblast. While most of the chromaffin cell precursors have very smalI cytoplasmic granules, a few The Adrenal in the Newborn Opossum (73-mm Snout-Rump) The adrenal anlagen can be visualized by scanning electron microscopy in the newborn (Fig. 4).Examination of the posterior abdominal wall of the newborn opossum shows that the rostral pole of the left adrenal anlage is located at the same transverse level as the rostral pole of the mesonephros. Medially, the entire left adrenal anlage borders the root of the mesentery, whereas laterally the rostral portion borders the mesonephros and the caudal portion is adjacent to the metanephros. At this stage the right adrenal is mostly hidden under the liver but can be seen adjacent to the inferior vena cava. The lateral, caudal portion borders the metanephros. Fig. 12.The adrenal gland is at the left and extra-adrenal chromaffin tissue (EA) is shown in the upper right-hand corner. A large number of chromaffin cells containing dark-staining argentaffn granules are seen near CR. C, cortex; N, nerves. Ammoniacal silver. X 180. Fig. 13. The chromaffh cells in the cord labeled CR have cytoplasm that is packed with dark-staining argentaffn granules. An arrow extending from CR crosses a cord of cortical cells and ends on a cord of chromaffin cells that have light-staining nonargentaffin cytoplasmic granules. C, cortical cell. Ammoniacal silver. x750. Fig. 14. Serial section through the adrenal gland of a 6-day-old opossum. Development of the medulla N)and distinct cortical zones such as the zona glomerulosa (GI begins to become obvious at this stage. C, cortical cells. Ammoniacal silver, ~ 2 2 0 . S.W. CARMICHAEL ET AL. 2 16 Fig. 15. Section of the adrenal gland of a 12-day-old opossum. Comparison of this figure with Figure 1 suggests that by day 12 the basic organization of the adult adrenal is obtained. G, zonu glomerdosa; F, zona fasciculuta; R, mna reticuluris; M, medulla. H and E. X 117. Fig. 16. Ultrastructure of a primitive sympathetic cell (PSC). Polyribosomes are densely distributed. Chromaffin vesicles (small arrow) and GER (large arrow) are sparse. G, Golgi complex; CI, cilium. ~7,990. have dense granules that stain intensely with toluidine blue. Serial sections stained with ammoniacal silver reveal that these granules have a positive argentaffin reaction (Fig. 6). Stromal constituents of the adrenal anlage include connective-tissue cells and fibers, unmyelinated nerves, Schwann cells, endothelial cells, and a few sinusoids. Unmyelinated nerve fibers enter the adrenals along the dorsomedial border where there are continuities between extra- and intra-adrenal chromaffin tissue (Fig. 6). A loose connective-tissue capsule is present, and the capsule along the ventromedial border of the right adrenal is fused with the adventitia of the inferior vena cava. nent, and a larger proportion of' the cells have argentaffin-positive cytoplasmic granules in their cytoplasm. The pheochromoblast is the predominant cell type from the third through the seventh postnatal days. During the third, fourth, and fifth days of postnatal development, the adrenal glands begin to segregate into cortical and medullary regions. By the third day the entrance of nerve fibers and the continuity between extra- and intra-adrenal chromaffin tissue is restricted to the hilus. Peripheral borders of the gland, excluding the hilus, are composed of a layer of cortical cells. Intermingled cortical and chromaffin cells still form columns in the central region of 3- and 4-day-old adrenal glands (Fig. 12). By the fifth day there are columns in the center of the gland that consist of only chromaffin cells (Fig. 13). At this stage, cells demonstrating an intense argentaffin reaction can be seen in addition to the morphologically similar, but nonargentaffin, cells. Both argentaffin-positive and -negative cells have cytoplasm divided into paranuclear Golgi regions and peripheral granular regions. Morphological maturation of the adrenal gland appears to take place between postnatal days 6 and 12. Zonation of the cortex begins to become distinct by day 6 (Fig. 14). The adrenal medulla becomes more distinct, and cords of chromaffin cells begin to form in the medulla so that it resembles the morphology seen in mature opossums. By the eighth day, most of the chromaffin cells appear to be mature. Although most of the cells in The Adrenal in Older Neonates During the next 6 days after birth, the adrenal glands grow conspicuously larger and change in relative position as the metanephros continues to develop and the mesonephros and gonadal primordia recede caudally. By the eighth postnatal day, the adrenals are in their definitive position medial to the metanephric kidneys (Fig. 10). The most obvious difference in the histology of the adrenal gland at 2 days is the conspicuous increase in the size and number of sinusoids. The sinusoids appear to arrange the parenchyma into anastomosing columns. Most columns contain both chromaffin and cortical cells (Fig. 11).The amount of chromaffin tissue, both intraand extra-adrenal, is greater than that observed on the preceding day. Argentaffin reactions are more promi- POSTNATAL DEVELOPMENT OF OPOSSUM ADRENAL MEDULLA the medulla are chromaffin cells, a few cords of cortical cells persist. A continuity between intra- and extra-adrenal chromaffin tissue persists at the hilus. By day 12, the zonation of the cortex is distinct, and the medulla is clearly formed (Fig. 15). DISCUSSION The opossum (Didelphis uirginiana) is a unique mammal in that it is the only marsupial found naturally in North America. Furthermore, it is considered to be a primitive or generalized mammal (Colbert, 1967). The marsupial characteristic of some embryonic and all fetal development occurring postnatally, in addition to the fact that the opossum is a generalized marsupial, makes this mammal an ideal model for the study of mammalian development (Jurgelski, 1974). This study shows that the adult opossum adrenal medulla is typical €or mammals. The gross anatomical position of the adrenal gland is typical, although the right gland is more dorsal to the inferior vena cava than is commonly seen. The central aggregation of chromaffin cells to form the medulla of the gland is the typical mammalian pattern and is quite distinct from the avian and amphibian patterns. This factor becomes important when the development of the adrenal medulla in the opossum and lower vertebrates is compared. Two cell types are observed in the adrenal medulla of the adult opossum following preparation with glutaraldehyde and osmium (Coupland et al., 1964)and staining of plastic sections with toluidine blue. Cells with dark cytoplasmic granules represent norepinephrine-containing cells and those with light granules represent epinephrine-containing cells. These two catecholamines are present in about equal amounts in the opossum adrenal, as shown by fluorometric analysis (see following paper). This is not the typical mammalian ratio, which is about 90% epinephrine in adult specimens (West, 1955),but it is similar to the cat, which has been used to advantage in some experiments because of the 1:l ratio of epinephrine and norepinephrine (Robinson et al., 1983). Studies on other mammals have shown that the proliferation of the adrenal cortical anlage from the coelomic mesoderm is the initial indication that adrenal development has begun. The adrenal medulla begins to form following the migration of undifferentiated precursors of chromaffin cells into the adrenal cortical anlage (Bourne, 1949). This migration is preceded by stages that influence the determination of the cells while directing them toward the anlage. Following the migration of cells into the anlage, poorly understood interactions direct the formation of this gland, which is unique to mammals. The stages and timing of the normal mammalian adrenal medulla development will be compared to those observed in the opossum. Soon after the cortical anlage is first recognized, most studies have identified small, intensely staining basophilic cells migrating through the mesoderm at a prevertebral position. These cells have been named primitive sympathetic cells by Coupland (19651, and he considered them to be the migratory totipotent precursors of definitive chromaffin cells and their intermediate form. In an elegant study on birds, LeDouarin and Teillet (1974) showed that these cells are of ectodermal neural crest origin. Neural crest cells, which are to become primitive sympathetic cells, are induced to undergo 217 adrenergic cell differentiation by their associations during migration with the ventral neural tube and somites (Norr, 1973).It is thought that before these cells undergo differentiation and proliferation they must encounter a maintenance factor in the extracellular matrix of the dorsal trunk mesoderm. This unidentified factor seems to be concentrated in the area of the adrenal anlage (LeDouarin and Teillet, 1974).The neural crest cells and their progeny are thought to be directed along preferential pathways in the intersomitic mesoderm toward the adrenal anlage (LeDouarin and Teillet, 1974). We found that cells fitting the description of primitive sympathetic cells are located in a position that is lateral and ventral to the aorta (Fig. 6). At a later stage, these cells are shown to form part of a transitory mass of precursor cells adjacent to the medial aspect of the cortical anlage (Fig. 12). It should be emphasized that a number of studies on mammalian embryos have shown sympathetic cell migration and formation of a mass of precursor cells medial to the developing adrenal anlage while the embryo is in utero. In the opossum, the same typical stages which precede actual adrenal medulla formation take place postnatally. The initiation of the formation of the adrenal medulla is marked by the invasion of the cortical anlage by cells of the precursor cell mass. It is interesting that among the mammalian species studied, there is a considerable variation in the age of embryos when this invasion of the anlage is taking place. On the other hand, there is a remarkable consistency throughout the species reviewed with regard to the size of the developing fetuses at this stage. This includes studies of the rabbit (Mitsukuri, 1882; Kohn, 1903), cat (Davies, 1937), guinea pig (Harmon and Derbyshire, 1932), insectivores (Adams, 1958), rat (Pankratz, 1931; Elfvin, 1967; Daikoku et al., 1969), mouse (Miller, 1926; Waring, 1936; Fernholm, 1971; Theiler and Muntener, 1974), pig (Stadnicka and Van Wynsberghe, 19821, and man (Keene et al., 1927; Crowder, 1957; Coupland, 1952, 1965; Hervonen, 1971). Our data from the opossum agree with these studies; thus, it may be important to note that this critical phase in the development of the mammalian adrenal medulla is initiated in fetuses of a characteristic size (14-20 mm crown-rump length). This size also may be related to other embryonic events such as closure of a neural tube or an increased vascularization of the posterior abdominal wall. Studies on other mammals demonstrate that the embryos reach this characteristic length in utero. Our observations, however, present a mammal which is an exception in that this critical phase is reached when the animal is extrauterine. The opossum does not obtain the length characteristic of embryos initiating adrenal medullary development until after its premature birth. During the period in the opossum that primitive sympathetic cells are forming a mass adjacent to the medial aspect of the developing anlage, another cell type becomes conspicuous. This cell (Fig. 16) has a larger, less basophilic nucleus, a moderate amount of chromatin, and more cytoplasm than the primitive cell. This cell resembles the pheochromoblast described by Coupland (1965). Pheochromoblasts have also been identified within the developing adrenal medulla in the rat (Elfvin, 1967; Ratzenhofer and Muller, 1967) and the chick (Fujita et al., 1976). In the next stage the precursor cells begin to become S.W. CARMICHAEL ET AL. 2 18 localized in the central region of the gland (Fig. 14). During this stage the medullary cells become directly associated with sinusoids. As this is occurring, definitive chromaffin cells begin to appear in the center of the gland. These chromaffin cells fit the description given by Coupland (1965); their most characteristic feature is a n extremely granular cytoplasm. The differentiated chromaffin cells become organized in cordlike arrangement along the sinusoids in the center of the gland. This observation would suggest that chromaffin cell differentiation is linked to vascular development. Differentiation could also be associated with a maintenance factor in the intercellular tissue fluid (LeDouarin and Teillet, 1974). The adrenal gland in the opossum completes its development in a fashion similar to that described in other mammals (Fig. 15). In other studies, the essentially adult organization of the adrenal medulla is complete by birth. This has been shown to be the case in rabbit (Coupland and Weakley, 1968) and rat (Elfvin, 1967). This is also true in human specimens; however, Coupland (1952) reported that precursor cells can be identified within the adrenal medulla up to the end of the first postnatal year. This study has established that the adrenal medulla in the opossum develops similarly t o that of other mammals. Since the opossum is a primitive mammal, this points out commonality in the development of this organ. The most unique feature is that the entrance of pheochromoblasts into the cortical anlage and the development of chromaffin cells occur postnatally in the opossum, in contrast to development in utero for all other mammals studied. Therefore, we propose the adrenal medulla of the opossum as a developmental model since it develops postnally and with a typical mammalian histological pattern. This model presents the advantage of being accessible to physical, chemical, or operative manipulations in a known, quantifiable manner. 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