383
J. Anat. (1977), 124, 2, pp. 383-391 With 6 figures Printed in Great Britain
Formation of concentric whorls of rough endoplasmic reticulum in the adrenal gland of the Mongolian gerbil PETER A. NICKERSON
Department of Pathology, State University of New York at Buffalo, 180 Race Street, Buffalo, New York 14207, U.S.A.
(Accepted 20 August 1976) INTRODUCTION
Nickerson & Curtis (1969) first reported concentric whorls of rough endoplasmic reticulum in adrenocortical cells of the Mongolian gerbil. The whorls occur exclusively within cells located at the border between the zona fasciculata and zona reticularis (border region). Cells with whorls have some features of the zona fasciculata, such as lipid droplets and moderate amounts of smooth endoplasmic reticulum; the border cells also have numerous lysosomes and small mitochondria characteristic of zona reticularis cells (Nickerson, 1971). The function of cells in the border region is not fully understood. Nickerson (1970) has postulated that the region is a source of reserve cells which may transform into actively secreting steroidogenic cells under appropriate stimulation. Stress (Nickerson, 1972) or ACTH (Nickerson, 1970) cause transformation of whorls of rough endoplasmic reticulum into focal areas of smooth endoplasmic reticulum, a characteristic of cells actively engaged in steroidogenesis (Blanchette, 1966; Christensen & Fawcett, 1966). It was the purpose of the present study to determine when and where the whorls of rough endoplasmic reticulum first appeared in development. MATERIALS AND METHODS
Eight pregnant Mongolian gerbils (Meriones unguiculatus) were obtained from Tumblebrook Farms, North Brookfield, Massachusetts. Two animals were killed after 18 days' gestation, and the other animals were allowed to deliver. Three males and three females were killed on the day of birth and at 6, 21,28, 35 and 42 days after birth respectively. Animals were weaned at 28 days and placed in individual cages. Animals from birth onwards were killed by decapitation. The right adrenal gland was removed quickly and placed in 3 % purified glutaraldehyde (Ladd Research Industries, Burlington, Vermont) buffered to pH 7-4 with 01 M phosphate. Slices about 1 mm thick were fixed for 4 hours at 4 'C, then rinsed overnight in several changes of 0 1 M phosphate buffer (pH 7-4). In the case of fetal animals the peritoneal cavity was opened and the entire animal fixed in glutaraldehyde. The sex of the animals was ascertained, and the right adrenal gland removed, while working at the 3 x magnification of an American Optical dissecting microscope. Radial sections were cut from the adrenal gland with a microscalpel while the tissue was in 0-1 M phosphate buffer (pH 74) (Nickerson, 1970). The radial section included the capsule, zona glomerulosa, inner cortex and edge of the medulla. Tissue was post-fixed with 1 % osmium tetroxide in 0-1 M phosphate buffer 25-2
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(pH 7 2) and processed for electron microscopy (Nickerson, 1971). One micron sections were cut from all blocks and stained with toluidine blue to verify the zonal position of the tissue. Thin sections were cut with glass knives on a Porter-Blum MT-l ultramicrotome and stained with uranyl acetate (Stempak & Ward, 1964) and lead citrate (Reynolds, 1963) before examination with a Siemens 101 electron microscope. The left adrenal glands were processed for light microscopy by conventional techniques and 4 pm sections were stained with haematoxylin and eosin. One adrenal gland from each of eight animals either 35 or 42 days old was weighed after fixation in 10 % phosphate-buffered formalin. Quantitative data were expressed as the mean ±SEM. RESULTS
The adrenal gland in fetal and newborn animals was white in colour and spheroid in shape; the gland was situated adjacent to the kidney, although it was not enclosed by the kidney capsule. In post-weanling gerbils the adrenal gland was ellipsoid in contour. On the left side the gland was located in a retroperitoneal position and was found further away from the kidney than in fetal and newborn animals. Conversely, on the right side, the adrenal gland was more closely connected to the kidney capsule by connective tissue. At 35 days the weight of the adrenal gland in females was 5-2 + 0 1 mg and in males 5 +± 0-1 mg (left adrenal gland), whereas at 42 days the weight in females was 6-7±03 mg and in males 5-6±0-1 mg. No x-zone similar to that in mice (Sato, 1968) was observed adjacent to the medulla in fetal, newborn, weanling or post-weanling animals. By light microscopy, cortical cells were seen arranged in branching cords with tortuous interconnecting sinusoids in fetal and newborn animals. No distinct cortical zones were observed by light microscopy until after weaning. In post-weanling animals (35 days) a distinct inner cortex was divided into a zona fasciculata and a zona reticularis. Adrenal ultrastructure The ultrastructure of the adrenal gland in male and female gerbils was identical at all stages examined and so there is no point in describing the sexes separately. Fetal adrenal gland Cells throughout the fetal cortex were identical and there was no distinct zonation. Mitochondria were circular in shape and cristae were tubular. The electron opacity of the mitochondrial matrix varied from moderate to dense. Focal areas or stacks of rough endoplasmic reticulum with fenestrated cisternae were observed in cells throughout the cortex (Fig. 1). The cisternae were frequently located in a paranuclear position. Many cisternae of smooth endoplasmic reticulum were scattered throughout the cytoplasm. The numbers of lipid droplets varied considerably; in one cell droplets might be numerous, whereas in an adjacent cell droplets might not Fig. 1. Fetal adrenal gland. Parallel stacks of fenestrated (arrow) rough endoplasmic reticulum are to be seen in the cytoplasm. Mitochondrial cristae are tubular. Smooth endoplasmic
reticulum (SER). x 16200. Fig. 2. Zona glomerulosa from newbom animal. A prominent Golgi apparatus (G) with numerous associated vesicles is observed in a paranuclear position. Mitochondrial cristae (arrow) are plate-like. x 22 400.
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PETER A. NICKERSON 386 be present at all (Fig. 1). In some cells lysosomes were closely apposed to lipid droplets. The Golgi apparatus was often aligned near the plasma membrane: numerous vesicles, many of which were coated, were associated with its cisternae.
Newborn animals Distinct zona glomerulosa cells were recognized at the periphery of the gland beneath the capsule on the day of birth (Fig. 2). Mitochondria in zona glomerulosa cells were small, and the cristae appeared either tubular or shelf-like; the mitochondrial matrix was electron-opaque. The Golgi complex was prominent and usually located in a juxtanuclear position. Endoplasmic reticulum was predominantly smooth-surfaced, although several cisternae of rough endoplasmic reticulum were dispersed randomly throughout the cytoplasm. The number of lipid droplets varied from cell to cell. No distinct zonation of the inner cortex was evident. Mitochondria in the inner cortex were elongate, and cristae were shelf-like (Fig. 3). Cells were larger and lipid droplets more numerous than in the zona glomerulosa. Numerous cisternae of smooth endoplasmic reticulum were scattered throughout the cell. Occasionally, small membrane-bound, electron-opaque bodies were aggregated in focal areas of the cytoplasm bordering the plasma membrane. 6, 28 days By 6 days after birth distinct zona reticularis cells were observed adjacent to the medulla, although the cells were not arranged in branching cords. Many of the mitochondria were especially large, and the mitochondrial cristae often assumed whorl-like configurations. Numerous lysosomes were present in the cells. Although endoplasmic reticulum was predominantly smooth-surfaced, there were occasional cistemae of rough endoplasmic reticulum. Lipid droplets were numerous in all inner cortical cells. Mitochondria in zona fasciculata cells were spherical and their cristae were tubular. Endoplasmic reticulum was smooth-surfaced and interspersed among numerous large, lipid droplets. The zona glomerulosa was identical in structure to that described above except that lipid droplets were particularly numerous. At weaning (28 days after birth) the cells in the zona glomerulosa and zona fasciculata (Fig. 4) were similar to those described at 6 days. Only very few cistemae of rough endoplasmic reticulum were observed in cells of the zona reticularis. Mitochondria were larger in some reticularis cells; interconnected, focal areas of smooth endoplasmic reticulum were present in the cytoplasm. Post-weaning (35, 42 days) At 35 and 42 days the zona glomerulosa did not differ from that described previously. Similarly, zona fasciculata cells at both times were identical in structure to that described at 6 days. By 42 days zona reticularis cells in general contained Fig. 3. Inner cortical cells from newborn animal. Numerous lipid droplets (L) are observed in the cytoplasm. Endoplasmic reticulum (SER) is smooth-surfaced. Mitochondria are elongate with tubular to plate-like cristae. x 24000. Fig. 4. Zona fasciculata cell from weanling gerbil (28 days). Large lipid droplets (L) are observed. Mitochondria contain tubular cristae. Smooth endoplasmic reticulum (SER). x 26400.
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J--.;.,I
SER
6
389 Formation of RER whorls few lipid droplets, and a smaller number of cisternae of smooth endoplasmic reticulum, than at previous times, although there was a fair amount of variation between cells. Lysosomes were prominent in zona reticularis cells at 42 days. Concentric whorls of rough endoplasmic reticulum were first demonstrable in cells of the border region between the zona reticularis and zona fasciculata at 42 days (Fig. 5). Whorls were not present in cells in the inner zona reticularis, zona fasciculata, or zona glomerulosa. In its whorled form the rough endoplasmic reticulum gave rise to a concentric spheroid structure (Fig. 5): alternatively, elongate, parallel cisternae coursed throughout the cytoplasm (Fig. 6). The whorls were often interspersed among abundant cisternae of smooth endoplasmic reticulum. Many lysosomes were observed within cells of the border region. DISCUSSION
Concentric whorls of rough endoplasmic reticulum occur in cells located in the region bordering the zona fasciculata-zona reticularis in young adult Mongolian gerbils (Nickerson & Curtis, 1969). It is of special interest that the whorls first appear at 42 days after birth in the same region as they are found in adults. Whorls of rough reticulum do not appear in other cells of the cortex. The whorls develop in cells virtually filled with smooth endoplasmic reticulum. Abundant smooth endoplasmic reticulum is characteristic of actively secreting steroid cells (Christensen & Fawcett, 1966; Blanchette, 1966) and may well reflect an active synthesis of cortisol (Oliver & Peron, 1964). The smooth reticulum contains enzymes involved in the synthesis of cholesterol (Chesterton, 1968), the conversion of pregnenolone to progesterone (Beyer & Samuels, 1956) and of progesterone to deoxycorticosterone (Ryan & Engel, 1957). Once the whorls are formed the smooth endoplasmic reticulum in border cells obviously decreases because little smooth reticulum is observed in border cells of young adult gerbils (Nickerson, 1971). In all probability the decreased amount of smooth endoplasmic reticulum reflects less active steroidogenesis. The function of the cells containing whorls is unclear, although they may possibly serve as reserve cells which can, upon demand, transform into actively secreting steroid cells. Nickerson (1972) has shown that concentric whorls of rough endoplasmic reticulum in border cells disappear following stress or exogenous ACTH (Nickerson, 1970) and are replaced by focal areas of smooth reticulum. It is of interest that smooth endoplasmic reticulum is formed from rough endoplasmic reticulum (Dallner, Siekevitz & Palade, 1966). Conversely, procedures which abolish (hypophysectomy) or reduce (testosterone, dexamethasone) the secretion of ACTH leave the whorls unchanged (Nickerson, 1972). The border region is located between the zona fasciculata and zona reticularis. The zona fasciculata has abundant smooth endoplasmic reticulum, lipid droplets and numerous mitochondrial cristae (Nickerson, 1971.) On the other hand, the zona Fig. 5. Cells in border region from 42 days old gerbil. A concentric whorl of rough endoplasmic reticulum is observed in a paranuclear position. Numerous tubules of smooth endoplasmic reticulum (SER) are dispersed throughout the cytoplasm. Lipid droplet (L). x 22400. Fig. 6. Cells in border region from 42 days old gerbil. Parallel cistemnae of rough endoplasmic reticulum (ER) are found in a paranuclear location. Smooth endoplasmic reticulum (SER). x 17600.
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reticularis contains little smooth endoplasmic reticulum, few lipid droplets and small mitochondria with few cristae (Nickerson, 1971). The zona fasciculata is active in steroid synthesis whereas the function of the zona reticularis is less clear. Blackman (1946) considered the zona reticularis to be a source of sex steroids in man, but others have considered it to be a region of cell degeneration (Hoerr, 1931). Whorls form after the zona fasciculata and zona reticularis become fully differentiated. Distinct zona reticularis cells were identified by electron microscopy before branching cords and prominent sinusoids could be seen by light microscopy. In the mouse (Muntener & Theiler, 1974) and rat (Mitchell, 1948), the zona reticularis develops 3 weeks after birth. Sucheston & Cannon (1968) observed a poorly organized zona reticularis during the second year in children; permanent characteristics of the zona reticularis appeared by the eleventh to twelfth year. The Mongolian gerbil differs from some other animals in lacking a fetal cortex or x-zone. A transient x-zone, or juxtamedullary zone, occurs in fetal and newborn mice (Sato, 1968) whereas a fetal cortex is observed in man (McNutt & Jones, (1970) and guinea-pig (Black, 1972). A distinct zona glomerulosa was observed in the gerbil at birth. Lever (1956) also observed a distinct zona glomerulosa in newborn rats and Nussdorfer (1970) found that the ultrastructure of the zona glomerulosa closely resembled that in adult animals. The fetal adrenal gland in the Mongolian gerbil contained fenestrated cisternae of rough endoplasmic reticulum in all cortical cells; these structures had disappeared by the time of birth. Similar structures occur in the human fetal adrenal gland (McNutt & Jones, 1970), but they persist into adult life (Long & Jones, 1967). SUMMARY
All cortical cells appeared alike in fetal gerbils of both sexes though glomerulosa cells were distinguishable at the time of birth. Fenestrated cisternae of rough endoplasmic reticulum were observed in all cortical cells of fetal gerbils, but smooth endoplasmic reticulum predominated. Concentric whorls of rough endoplasmic reticulum first appeared in adrenocortical cells at 42 days of age. The whorls developed within cells having abundant smooth endoplasmic reticulum and which were located in the region between the zona fasciculata and zona reticularis. Cells with whorls possibly serve as reserve cells which transform into actively secreting steroid cells in times of stress.
This investigation was supported by research grant HL 06975 from the National Heart and Lung Institute. The author is grateful to Mrs Neonile Fylypiw, Mr Luther Joseph, Mrs Geneva Joseph, Mrs Elisabeth Lawson and Mr Robert Linsmair for skilled technical assistance. REFERENCES BEYER, K. F. & SAMUELS, L. T. (1956). Distribution of steroid-3-,8-ol-dehydrogenase in cellular structures of the adrenal gland. Journal of Biological Chemistry 219, 69-76. BLACK, V. H. (1972). The development of smooth-surfaced endoplasmic reticulum in adrenal cortical cells of fetal guinea pigs. American Journal of Anatomy 135, 381-418. BLACKMAN, S. S. (1946). Concerning the function and origin of the reticular zone of the adrenal cortex. Johns Hopkins Hospital Bulletin 78, 180-208. BLANCHEI-rE, E. J. (1966). Ovarian steroid cells. II. The lutein cell. Journal of Cell Biology 31, 517-542. CHESTERTON, C. J. (1968). Distribution of cholesterol precursors and other lipids among rat liver intracellular structures. Journal ofBiological Chemistry 243. 1147-1151.
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CHRISTENSEN, A. K. & FAWCETT, D. W. (1966). The fine structure of testicular interstitial cells in mice. American Journal of Anatomy 118, 551-572. DALLNER, G., SIEKEVITZ, P. & PALADE, G. E. (1966). Biogenesis of endoplasmic reticulum membranes. I. Structural and chemical differentiation in developing rat hepatocyte. Journal of Cell Biology 30, 73-96. HOERR, N. (1931). The cells of the suprarenal cortex in the guinea pig. Their reaction to injury and their replacement. American Journal of Anatomy 48, 139-197. LEVER, J. D. (1956). Adreno-cortical histogenesis in the rat: with observations on lipid and ascorbic acid distribution. Journal of Anatomy 89, 293-300. LONG, J. A. & JONES, A. L. (1967). Observations on the fine structure of the adrenal cortex of man. Laboratory Investigation 17, 355-370. McNuTr, N. S. & JONES, A. L. (1970). Observations on the ultrastructure of cyto-differentiation in the human fetal adrenal cortex. Laboratory Investigation 22, 513-527. MITCHELL, R. M. (1948). Histological changes and mitotic activity in the rat adrenal during postnatal development. Anatomical Record 101, 161-185. MCUNTNER, M. & THEILER, K. (1974). Die Entwicklung der Nebennieren der Maus. II. Postnatale Entwicklung. Zeitschrift fur Anatomie und Entwicklungsgeschichte 144, 205-214. NICKERSON, P. A. (1970). Effects of ACTH on membranous whorls in the adrenal gland of the Mongolian gerbil. Anatomical Record 166, 479-490. NICKERSON, P. A. (1971). Fine structure of the Mongolian gerbil adrenal cortex. Anatomical Record 171, 443-456. NICKERSON, P. A. (1972). Effect of testosterone, dexamethasone, and hypophysectomy on membranous whorls in the adrenal gland of the Mongolian gerbil. Anatomical Record 174, 191-204. NICKERSON, P. A. & CURTIS, J. C. (1969). Concentric whorls of rough endoplasmic reticulum in adrenocortical cells of the Mongolian gerbil. Journal of Cell Biology 40, 859-862. NUSSDORFER, G. G. (1970). The fine structure of the newborn rat adrenal cortex. 1. Zona glomerulosa and zona fasciculata. Zeitschrift fur Zellforschung und mikroskopische Anatomie 103, 382-397. OLIVER, J. T. & PERON, F. G. (1964). 19-hydroxy-1 1-deoxycortisol, a major steroid secreted by the adrenal gland of the Mongolian gerbil. Steroids 4, 351-363. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17, 208-212. RYAN, K. J. & ENGEL, L. L. (1957). Hydroxylation of steroids at carbon 21. Journal of Biological Chemistry 225. 103-114. SATO, T. (1968). The fine structure of the mouse adrenal x zone. Zeitschrift fur Zellforschung und mikroskopische Anatomie 87, 315-329. STEMPAK, J. C. & WARD, R. T. (1964). An improved staining method for electron microscopy. Journal of Cell Biology 22, 697-701. SUCHESTON, M. E. & CANNON, M. S. (1968). Development of zonular pattems in the human adrenal gland. Journal of Morphology 126, 477-492.
J. Anat. (1977), 124, 2, pp. 383-391 With 6 figures Printed in Great Britain
Formation of concentric whorls of rough endoplasmic reticulum in the adrenal gland of the Mongolian gerbil PETER A. NICKERSON
Department of Pathology, State University of New York at Buffalo, 180 Race Street, Buffalo, New York 14207, U.S.A.
(Accepted 20 August 1976) INTRODUCTION
Nickerson & Curtis (1969) first reported concentric whorls of rough endoplasmic reticulum in adrenocortical cells of the Mongolian gerbil. The whorls occur exclusively within cells located at the border between the zona fasciculata and zona reticularis (border region). Cells with whorls have some features of the zona fasciculata, such as lipid droplets and moderate amounts of smooth endoplasmic reticulum; the border cells also have numerous lysosomes and small mitochondria characteristic of zona reticularis cells (Nickerson, 1971). The function of cells in the border region is not fully understood. Nickerson (1970) has postulated that the region is a source of reserve cells which may transform into actively secreting steroidogenic cells under appropriate stimulation. Stress (Nickerson, 1972) or ACTH (Nickerson, 1970) cause transformation of whorls of rough endoplasmic reticulum into focal areas of smooth endoplasmic reticulum, a characteristic of cells actively engaged in steroidogenesis (Blanchette, 1966; Christensen & Fawcett, 1966). It was the purpose of the present study to determine when and where the whorls of rough endoplasmic reticulum first appeared in development. MATERIALS AND METHODS
Eight pregnant Mongolian gerbils (Meriones unguiculatus) were obtained from Tumblebrook Farms, North Brookfield, Massachusetts. Two animals were killed after 18 days' gestation, and the other animals were allowed to deliver. Three males and three females were killed on the day of birth and at 6, 21,28, 35 and 42 days after birth respectively. Animals were weaned at 28 days and placed in individual cages. Animals from birth onwards were killed by decapitation. The right adrenal gland was removed quickly and placed in 3 % purified glutaraldehyde (Ladd Research Industries, Burlington, Vermont) buffered to pH 7-4 with 01 M phosphate. Slices about 1 mm thick were fixed for 4 hours at 4 'C, then rinsed overnight in several changes of 0 1 M phosphate buffer (pH 7-4). In the case of fetal animals the peritoneal cavity was opened and the entire animal fixed in glutaraldehyde. The sex of the animals was ascertained, and the right adrenal gland removed, while working at the 3 x magnification of an American Optical dissecting microscope. Radial sections were cut from the adrenal gland with a microscalpel while the tissue was in 0-1 M phosphate buffer (pH 74) (Nickerson, 1970). The radial section included the capsule, zona glomerulosa, inner cortex and edge of the medulla. Tissue was post-fixed with 1 % osmium tetroxide in 0-1 M phosphate buffer 25-2
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(pH 7 2) and processed for electron microscopy (Nickerson, 1971). One micron sections were cut from all blocks and stained with toluidine blue to verify the zonal position of the tissue. Thin sections were cut with glass knives on a Porter-Blum MT-l ultramicrotome and stained with uranyl acetate (Stempak & Ward, 1964) and lead citrate (Reynolds, 1963) before examination with a Siemens 101 electron microscope. The left adrenal glands were processed for light microscopy by conventional techniques and 4 pm sections were stained with haematoxylin and eosin. One adrenal gland from each of eight animals either 35 or 42 days old was weighed after fixation in 10 % phosphate-buffered formalin. Quantitative data were expressed as the mean ±SEM. RESULTS
The adrenal gland in fetal and newborn animals was white in colour and spheroid in shape; the gland was situated adjacent to the kidney, although it was not enclosed by the kidney capsule. In post-weanling gerbils the adrenal gland was ellipsoid in contour. On the left side the gland was located in a retroperitoneal position and was found further away from the kidney than in fetal and newborn animals. Conversely, on the right side, the adrenal gland was more closely connected to the kidney capsule by connective tissue. At 35 days the weight of the adrenal gland in females was 5-2 + 0 1 mg and in males 5 +± 0-1 mg (left adrenal gland), whereas at 42 days the weight in females was 6-7±03 mg and in males 5-6±0-1 mg. No x-zone similar to that in mice (Sato, 1968) was observed adjacent to the medulla in fetal, newborn, weanling or post-weanling animals. By light microscopy, cortical cells were seen arranged in branching cords with tortuous interconnecting sinusoids in fetal and newborn animals. No distinct cortical zones were observed by light microscopy until after weaning. In post-weanling animals (35 days) a distinct inner cortex was divided into a zona fasciculata and a zona reticularis. Adrenal ultrastructure The ultrastructure of the adrenal gland in male and female gerbils was identical at all stages examined and so there is no point in describing the sexes separately. Fetal adrenal gland Cells throughout the fetal cortex were identical and there was no distinct zonation. Mitochondria were circular in shape and cristae were tubular. The electron opacity of the mitochondrial matrix varied from moderate to dense. Focal areas or stacks of rough endoplasmic reticulum with fenestrated cisternae were observed in cells throughout the cortex (Fig. 1). The cisternae were frequently located in a paranuclear position. Many cisternae of smooth endoplasmic reticulum were scattered throughout the cytoplasm. The numbers of lipid droplets varied considerably; in one cell droplets might be numerous, whereas in an adjacent cell droplets might not Fig. 1. Fetal adrenal gland. Parallel stacks of fenestrated (arrow) rough endoplasmic reticulum are to be seen in the cytoplasm. Mitochondrial cristae are tubular. Smooth endoplasmic
reticulum (SER). x 16200. Fig. 2. Zona glomerulosa from newbom animal. A prominent Golgi apparatus (G) with numerous associated vesicles is observed in a paranuclear position. Mitochondrial cristae (arrow) are plate-like. x 22 400.
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PETER A. NICKERSON 386 be present at all (Fig. 1). In some cells lysosomes were closely apposed to lipid droplets. The Golgi apparatus was often aligned near the plasma membrane: numerous vesicles, many of which were coated, were associated with its cisternae.
Newborn animals Distinct zona glomerulosa cells were recognized at the periphery of the gland beneath the capsule on the day of birth (Fig. 2). Mitochondria in zona glomerulosa cells were small, and the cristae appeared either tubular or shelf-like; the mitochondrial matrix was electron-opaque. The Golgi complex was prominent and usually located in a juxtanuclear position. Endoplasmic reticulum was predominantly smooth-surfaced, although several cisternae of rough endoplasmic reticulum were dispersed randomly throughout the cytoplasm. The number of lipid droplets varied from cell to cell. No distinct zonation of the inner cortex was evident. Mitochondria in the inner cortex were elongate, and cristae were shelf-like (Fig. 3). Cells were larger and lipid droplets more numerous than in the zona glomerulosa. Numerous cisternae of smooth endoplasmic reticulum were scattered throughout the cell. Occasionally, small membrane-bound, electron-opaque bodies were aggregated in focal areas of the cytoplasm bordering the plasma membrane. 6, 28 days By 6 days after birth distinct zona reticularis cells were observed adjacent to the medulla, although the cells were not arranged in branching cords. Many of the mitochondria were especially large, and the mitochondrial cristae often assumed whorl-like configurations. Numerous lysosomes were present in the cells. Although endoplasmic reticulum was predominantly smooth-surfaced, there were occasional cistemae of rough endoplasmic reticulum. Lipid droplets were numerous in all inner cortical cells. Mitochondria in zona fasciculata cells were spherical and their cristae were tubular. Endoplasmic reticulum was smooth-surfaced and interspersed among numerous large, lipid droplets. The zona glomerulosa was identical in structure to that described above except that lipid droplets were particularly numerous. At weaning (28 days after birth) the cells in the zona glomerulosa and zona fasciculata (Fig. 4) were similar to those described at 6 days. Only very few cistemae of rough endoplasmic reticulum were observed in cells of the zona reticularis. Mitochondria were larger in some reticularis cells; interconnected, focal areas of smooth endoplasmic reticulum were present in the cytoplasm. Post-weaning (35, 42 days) At 35 and 42 days the zona glomerulosa did not differ from that described previously. Similarly, zona fasciculata cells at both times were identical in structure to that described at 6 days. By 42 days zona reticularis cells in general contained Fig. 3. Inner cortical cells from newborn animal. Numerous lipid droplets (L) are observed in the cytoplasm. Endoplasmic reticulum (SER) is smooth-surfaced. Mitochondria are elongate with tubular to plate-like cristae. x 24000. Fig. 4. Zona fasciculata cell from weanling gerbil (28 days). Large lipid droplets (L) are observed. Mitochondria contain tubular cristae. Smooth endoplasmic reticulum (SER). x 26400.
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J--.;.,I
SER
6
389 Formation of RER whorls few lipid droplets, and a smaller number of cisternae of smooth endoplasmic reticulum, than at previous times, although there was a fair amount of variation between cells. Lysosomes were prominent in zona reticularis cells at 42 days. Concentric whorls of rough endoplasmic reticulum were first demonstrable in cells of the border region between the zona reticularis and zona fasciculata at 42 days (Fig. 5). Whorls were not present in cells in the inner zona reticularis, zona fasciculata, or zona glomerulosa. In its whorled form the rough endoplasmic reticulum gave rise to a concentric spheroid structure (Fig. 5): alternatively, elongate, parallel cisternae coursed throughout the cytoplasm (Fig. 6). The whorls were often interspersed among abundant cisternae of smooth endoplasmic reticulum. Many lysosomes were observed within cells of the border region. DISCUSSION
Concentric whorls of rough endoplasmic reticulum occur in cells located in the region bordering the zona fasciculata-zona reticularis in young adult Mongolian gerbils (Nickerson & Curtis, 1969). It is of special interest that the whorls first appear at 42 days after birth in the same region as they are found in adults. Whorls of rough reticulum do not appear in other cells of the cortex. The whorls develop in cells virtually filled with smooth endoplasmic reticulum. Abundant smooth endoplasmic reticulum is characteristic of actively secreting steroid cells (Christensen & Fawcett, 1966; Blanchette, 1966) and may well reflect an active synthesis of cortisol (Oliver & Peron, 1964). The smooth reticulum contains enzymes involved in the synthesis of cholesterol (Chesterton, 1968), the conversion of pregnenolone to progesterone (Beyer & Samuels, 1956) and of progesterone to deoxycorticosterone (Ryan & Engel, 1957). Once the whorls are formed the smooth endoplasmic reticulum in border cells obviously decreases because little smooth reticulum is observed in border cells of young adult gerbils (Nickerson, 1971). In all probability the decreased amount of smooth endoplasmic reticulum reflects less active steroidogenesis. The function of the cells containing whorls is unclear, although they may possibly serve as reserve cells which can, upon demand, transform into actively secreting steroid cells. Nickerson (1972) has shown that concentric whorls of rough endoplasmic reticulum in border cells disappear following stress or exogenous ACTH (Nickerson, 1970) and are replaced by focal areas of smooth reticulum. It is of interest that smooth endoplasmic reticulum is formed from rough endoplasmic reticulum (Dallner, Siekevitz & Palade, 1966). Conversely, procedures which abolish (hypophysectomy) or reduce (testosterone, dexamethasone) the secretion of ACTH leave the whorls unchanged (Nickerson, 1972). The border region is located between the zona fasciculata and zona reticularis. The zona fasciculata has abundant smooth endoplasmic reticulum, lipid droplets and numerous mitochondrial cristae (Nickerson, 1971.) On the other hand, the zona Fig. 5. Cells in border region from 42 days old gerbil. A concentric whorl of rough endoplasmic reticulum is observed in a paranuclear position. Numerous tubules of smooth endoplasmic reticulum (SER) are dispersed throughout the cytoplasm. Lipid droplet (L). x 22400. Fig. 6. Cells in border region from 42 days old gerbil. Parallel cistemnae of rough endoplasmic reticulum (ER) are found in a paranuclear location. Smooth endoplasmic reticulum (SER). x 17600.
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reticularis contains little smooth endoplasmic reticulum, few lipid droplets and small mitochondria with few cristae (Nickerson, 1971). The zona fasciculata is active in steroid synthesis whereas the function of the zona reticularis is less clear. Blackman (1946) considered the zona reticularis to be a source of sex steroids in man, but others have considered it to be a region of cell degeneration (Hoerr, 1931). Whorls form after the zona fasciculata and zona reticularis become fully differentiated. Distinct zona reticularis cells were identified by electron microscopy before branching cords and prominent sinusoids could be seen by light microscopy. In the mouse (Muntener & Theiler, 1974) and rat (Mitchell, 1948), the zona reticularis develops 3 weeks after birth. Sucheston & Cannon (1968) observed a poorly organized zona reticularis during the second year in children; permanent characteristics of the zona reticularis appeared by the eleventh to twelfth year. The Mongolian gerbil differs from some other animals in lacking a fetal cortex or x-zone. A transient x-zone, or juxtamedullary zone, occurs in fetal and newborn mice (Sato, 1968) whereas a fetal cortex is observed in man (McNutt & Jones, (1970) and guinea-pig (Black, 1972). A distinct zona glomerulosa was observed in the gerbil at birth. Lever (1956) also observed a distinct zona glomerulosa in newborn rats and Nussdorfer (1970) found that the ultrastructure of the zona glomerulosa closely resembled that in adult animals. The fetal adrenal gland in the Mongolian gerbil contained fenestrated cisternae of rough endoplasmic reticulum in all cortical cells; these structures had disappeared by the time of birth. Similar structures occur in the human fetal adrenal gland (McNutt & Jones, 1970), but they persist into adult life (Long & Jones, 1967). SUMMARY
All cortical cells appeared alike in fetal gerbils of both sexes though glomerulosa cells were distinguishable at the time of birth. Fenestrated cisternae of rough endoplasmic reticulum were observed in all cortical cells of fetal gerbils, but smooth endoplasmic reticulum predominated. Concentric whorls of rough endoplasmic reticulum first appeared in adrenocortical cells at 42 days of age. The whorls developed within cells having abundant smooth endoplasmic reticulum and which were located in the region between the zona fasciculata and zona reticularis. Cells with whorls possibly serve as reserve cells which transform into actively secreting steroid cells in times of stress.
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