J. Anat. (1979), 129, 3, pp. 459477 With 31 figures Printed in Great Britain
459
Morphological observations on the metanephros in the postnatal opossum, Didelphis virginiana WILLIAM J. KRAUSE, J. HARRY CUTTS AND C. ROLAND LEESON Department of Anatomy, School of Medicine, University of Missouri, Columbia, Missouri
(Accepted 16 August 1978) INTRODUCTION
The gestation period of the opossum is remarkable short, lasting only 12+ days. The newborn young crawl unaided from the birth canal to the maternal marsupium (pouch) and attach to a nipple. Didelphis is very immature at birth, and further development, including both differentiation and growth, takes place in the pouch during the postnatal period. The opossum is born with a large, well-developed mesonephros that is composed of several nephron units (Krause, Cutts & Leeson, 1979). Structurally, the subunits of the mesonephric nephrons resemble those found in the metanephros, but lack a loop of Henle. The majority of the mesonephric nephrons are thought to be functional during the first week of postnatal life (Gersh, 1937). There may be some functional overlap between the mesonephros and metanephros early in the postnatal period. The present study reports the structural changes observed during the postnatal development of the metanephros in the opossum. MATERIALS AND METHODS
One hundred and twenty opossums (Didelphis virginiana) were used in the study. The pouch young opossums were divided into the following groups according to their snout-rump lengths: 1 4 (newborn), 2 5, 3 0, 3 5, 4 0, 4 5, 5 5, 6 5, 7 5, 8 5, 9 5, 10.0, 11 0, 13 0, 15.0, 19-0, 20-0, 22-0 and 28-0 cm. Age determinations were based on results presented by Cutts, Krause & Leeson (1978). Five adults also were used. The animals were killed by decapitation and, as quickly as possible, the metanephroi were removed and placed either in Bouin's solution or in 10 % buffered neutral formalin. In addition, the urinary systems of three animals from the first six groups were fixed whole for serial sectioning. The tissues were processed routinely for embedding in paraffin, sectioned at 6,um and the following staining procedures employed: haematoxylin and eosin, van Gieson, and periodic acid-Schiff (PAS), before and after treatment with saliva. Additional blocks of tissue were fixed for 4 hours at 0 'C in 3-5 % glutaraldehyde buffered in 0 1 M Sorensen's phosphate buffer to a pH of 7'4. The tissues were washed in the phosphate buffer and post-fixed for 2 hours at 0 'C in 10 % osmium tetroxide buffered in 0-1 M phosphate buffer. The specimens were then passed through a graded series of ethanol solutions prior to clearing in propylene oxide and embedding in Epon 812. Thin sections of this material were mounted on uncoated grids and 0021-8782/79/2828-6460 $02.00 © 1979 Anat. Soc. G.B. & I.
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stained with uranyl acetate and lead citrate (Reynolds, 1963). The thin sections were examined in a RCA EMU-3F electron microscope operated at 50 kV. Thick sections (1-3,um) of the Epon-embedded material, cut on a LKB pyramitome with glass knives, were stained with toluidine blue for light microscopy. Tissues for scanning electron microscopy were fixed as for transmission electron microscopy and then dehydrated in ethanol and transferred to amyl acetate prior to critical point drying with liquid CO2. The dried tissues were placed on spinner stubs and coated with a gold-palladium alloy to a depth of 20 nm in a vacuum evaporator. Specimens were viewed in a Cambridge Stereoscan Mark II electron microscope. RESULTS
The metanephros lies medial to a large, well-developed mesonephros, and its cranial pole is related to the forming adrenal cortex (Fig. 1). In the newborn opossum the metanephros is small, generally round in shape, and consists primarily of collecting tubules that divide dichotomously near the renal capsule (Figs. 1, 2). The collecting tubules and developing nephron units are separated by delicate connective tissue (Fig. 2). A definite nephronogenic zone lies immediately beneath the connective tissue of the renal capsule, and consists of two to three layers of irregularly shaped mesenchymal cells that stain deeply with toluidine blue. The nephrons are in different stages of maturation; those nearest the renal pelvis are more differentiated than those in the peripheral cortex. Near the capsule, the nephrons are very immature in appearance and are in the initial stages of differentiation (Figs. 2-6). Although the nephron units in the metanephros of the newborn opossum vary considerably in their degree of maturation, all appear to follow the same pattern of glomerular and tubular differentiation and development. In the initial stages of development the expanded branches of the collecting tubules (ampullae) that lie immediately beneath
Fig. 1. A scanning electron micrograph of the posterior abdominal wall showing the adrenal cortex (A), metanephros (mt), mesonephros (ms), gonad (g), colon (c), mesonephric (md) and Mullerian ducts (mu). Newborn opossum. x 50. Fig. 2. The newborn metanephros cut near the renal pelvis shows numerous collecting tubules (ct), occasional glomeruli (g) and differentiating nephrons (arrows). Haematoxylin and eosin. x 100. Fig. 3. The cortical region adjacent to the capsule shows the ampulla of a collecting tubule (large arrow) surrounded by a mass of nephronogenic cells. Note the mitotic activity within this group of cells. A portion of collecting tubule (ct) and the tubular components of an adjacent nephron also are shown. Newborn opossum. Epon 812. Toluidine blue. x 400. Fig. 4. A developing nephron from a newborn opossum. A portion of a collecting tubule (ct) is shown near the top of the figure. The region of the forming renal corpuscle shows flattened peripheral cells (small arrows) which form the parietal layer of Bowman's capsule, and an adjacent layer of cuboidal cells (the forming visceral layer). A capillary (large arrows) is shown entering the developing corpuscle at the left, adjacent to the tubular portion of the nephron. The capillary expands near the centre immediately subjacent to the surrounding visceral layer of Bowman's capsule. Epon 812. Toluidine blue. x 400. Fig. 5. A developing nephron showing a renal corpuscle that is more differentiated than the one depicted in Fig. 4. The capsular space is established and the visceral layer (arrows) continues to differentiate in relation to the forming glomerulus. Newborn opossum. Epon 812. Toluidine blue. x 400. Fig. 6. A renal corpuscle showing further differentiation than that illustrated in Figs. 4 and 5. The parietal layer, visceral layer, glomerular capillary loops, capsular space and urinary pole (arrow) are clearly shown. Newborn opossum. Epon 812. Toluidine blue. x 400.
462
W. J. KRAUSE, J. H. CUTTS AND C. R. LEESON
APOW... C, A
B
D
7
Fig. 7. A schematic representation of early nephronogenesis and the formation of a renal corpuscle in the opossum metanephros. Initially, branches of the dividing collecting tubule (ampullae) are surrounded by primitive mesenchymal cells that migrate from a nephronogenic zone located immediately beneath the renal capsule (A). Some of these cells form a thin doublelayered cap around the expanding branches of the collecting tubule as a loose, oval aggregate of cells forms between the collecting tubule and its branch (A, B). As a central cavity appears, a single layer of component cells is arranged with the long axis of its cells perpendicular to the lumen. The formed renal vesicle shows considerable mitotic activity as it elongates (C). Two indentations then appear and transform the elongate form into a blind-ended, S-shaped tubule (D). The lower limb of the tubule (that furthermost from the cortex or the branch of the collecting tubule) will form the renal corpuscle; the middle limb will become the proximal tubule; the upper limb will form the loop of Henle and the distal tubule. As the S-shaped form is established, [Continued on factng page
The metanephros of the postnatal opossum
463
the renal capsule become surrounded by nephronogenic cells (Figs. 3, 7), which coalesce to form a solid oval mass in relation to the ampulla and to the collecting tubule. A central cavity then appears and a single layer of columnar cells forms, with the long axes of the cells lying perpendicular to the lumen. Continued division of the cells of the newly formed renal vesicle results in the formation of an elongated tubule. As the tubule continues to elongate, two indentations occur and transform it into an S-shaped tubule (Figs. 2, 7). The lower limb of the S-shaped structure, which is furthest from the external surface of the kidney, will differentiate into the renal corpuscle. The cells of the lower limb begin to flatten, whereas epithelial cells on the opposite side of the same limb appear to enlarge slightly (Fig. 7). Simultaneously a small capillary penetrates the area between the lower and middle limbs of the S-shaped tubule to provide the vascular component of the forming renal corpuscle. The developing capillaries become intimately associated with the columnar epithelial cells that are continuous with those of the remainder of the tubule (Figs. 4-7). As the visceral layer of Bowman's capsule differentiates and develops around the invading vasculature, the cells on the opposite side of this portion of the tubule form the simple squamous epithelium of the parietal layer of Bowman's capsule (Fig. 7). The lumen at this end of the S-shaped tubule is the capsular space of the forming renal corpuscle. As the glomerular capillaries become established the parietal layer of Bowman's capsule appears to constrict around the original vascular point of entry to establish the vascular pole. The capsular space of the forming renal corpuscle remains continuous with the lumen of the remainder of the nephron unit, and forms the urinary pole (Figs. 6,7). Considerable mitotic activity is observed in the expanding proximal and distal limbs of the tubular component of the developing nephron. Communication between the developing nephron unit and the collecting tubule is established concomitant with the differentiation of the renal corpuscle (Fig. 7). Ultrastructurally, cells of the nephronogenic layer and of the aggregate near the expanding branch of the collecting tubule appear irregular in shape and are without any apparent organization. The cytoplasm is of considerable electron density and contains numerous polyribosomes (Fig. 8). Occasional profiles of granular endoplasmic reticulum and small, scattered mitochondria also are observed. Nuclei are irregular and contain prominent nucleoli. After the parietal and visceral layers of Bowman's capsule have been established, the podocytes which make up the visceral layer show a definite orientation along the developing capillaries (Figs. 4-6, 9, 10). The differentiating podocytes appear dome-like and uniform in shape (Figs. 5, 6, 9, 10), and that surface which is associated with the developing glomerular capillaries lies on a delicate basal lamina (Figs. 9, 10). The podocytes often show long microprojections extending from their surface, and occasional cilia are observed (Fig. 10). Cells of the visceral layer show an electrondense cytoplasm and numerous free polyribosomes. When compared to the cells of a capillary enters the space between the middle and lower limbs of the tubule (E) to become intimately associated with the epithelium of this limb (E, F). The tubular epithelium associated with the forming capillaries remains cuboidal to columnar, whereas cells composing the remainder of this limb flatten to form the parietal layer of Bowman's capsule (F, G, H). As these events occur the forming nephron joins the collecting tubule (E, F). The parietal layer expands and appears to constrict around the point of entry of the capillary, resulting in the formation of the vascular pole (G, H). The remaining tubular epithelium covering the forming capillaries undergoes marked morphological changes and forms the visceral (podocytic) layer of Bowman's capsule (G, H).
464
W. J. KRAUSE, J. H. CUTTS AND C. R. LEESON ._ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~AL
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459
Morphological observations on the metanephros in the postnatal opossum, Didelphis virginiana WILLIAM J. KRAUSE, J. HARRY CUTTS AND C. ROLAND LEESON Department of Anatomy, School of Medicine, University of Missouri, Columbia, Missouri
(Accepted 16 August 1978) INTRODUCTION
The gestation period of the opossum is remarkable short, lasting only 12+ days. The newborn young crawl unaided from the birth canal to the maternal marsupium (pouch) and attach to a nipple. Didelphis is very immature at birth, and further development, including both differentiation and growth, takes place in the pouch during the postnatal period. The opossum is born with a large, well-developed mesonephros that is composed of several nephron units (Krause, Cutts & Leeson, 1979). Structurally, the subunits of the mesonephric nephrons resemble those found in the metanephros, but lack a loop of Henle. The majority of the mesonephric nephrons are thought to be functional during the first week of postnatal life (Gersh, 1937). There may be some functional overlap between the mesonephros and metanephros early in the postnatal period. The present study reports the structural changes observed during the postnatal development of the metanephros in the opossum. MATERIALS AND METHODS
One hundred and twenty opossums (Didelphis virginiana) were used in the study. The pouch young opossums were divided into the following groups according to their snout-rump lengths: 1 4 (newborn), 2 5, 3 0, 3 5, 4 0, 4 5, 5 5, 6 5, 7 5, 8 5, 9 5, 10.0, 11 0, 13 0, 15.0, 19-0, 20-0, 22-0 and 28-0 cm. Age determinations were based on results presented by Cutts, Krause & Leeson (1978). Five adults also were used. The animals were killed by decapitation and, as quickly as possible, the metanephroi were removed and placed either in Bouin's solution or in 10 % buffered neutral formalin. In addition, the urinary systems of three animals from the first six groups were fixed whole for serial sectioning. The tissues were processed routinely for embedding in paraffin, sectioned at 6,um and the following staining procedures employed: haematoxylin and eosin, van Gieson, and periodic acid-Schiff (PAS), before and after treatment with saliva. Additional blocks of tissue were fixed for 4 hours at 0 'C in 3-5 % glutaraldehyde buffered in 0 1 M Sorensen's phosphate buffer to a pH of 7'4. The tissues were washed in the phosphate buffer and post-fixed for 2 hours at 0 'C in 10 % osmium tetroxide buffered in 0-1 M phosphate buffer. The specimens were then passed through a graded series of ethanol solutions prior to clearing in propylene oxide and embedding in Epon 812. Thin sections of this material were mounted on uncoated grids and 0021-8782/79/2828-6460 $02.00 © 1979 Anat. Soc. G.B. & I.
_tS_ptS# ,* =I 460 *
W. J.
KRAUSE, J.
i.c&'~ a -.
H. CUTTS AND C. R. LEESON
Ss,;vs..I
,*
A~~~~~~~~~~~~~~~~~~~~
41gU st~~~~~~~c
G :ff
s;t& ~~~~~g t d~t
it~~~~~~~~~C 0S t;t ; !' '~4
-I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ d~~~~~~~~~d
5
5.p, .
-X.
The metanephros of the postnatal opossum
461
stained with uranyl acetate and lead citrate (Reynolds, 1963). The thin sections were examined in a RCA EMU-3F electron microscope operated at 50 kV. Thick sections (1-3,um) of the Epon-embedded material, cut on a LKB pyramitome with glass knives, were stained with toluidine blue for light microscopy. Tissues for scanning electron microscopy were fixed as for transmission electron microscopy and then dehydrated in ethanol and transferred to amyl acetate prior to critical point drying with liquid CO2. The dried tissues were placed on spinner stubs and coated with a gold-palladium alloy to a depth of 20 nm in a vacuum evaporator. Specimens were viewed in a Cambridge Stereoscan Mark II electron microscope. RESULTS
The metanephros lies medial to a large, well-developed mesonephros, and its cranial pole is related to the forming adrenal cortex (Fig. 1). In the newborn opossum the metanephros is small, generally round in shape, and consists primarily of collecting tubules that divide dichotomously near the renal capsule (Figs. 1, 2). The collecting tubules and developing nephron units are separated by delicate connective tissue (Fig. 2). A definite nephronogenic zone lies immediately beneath the connective tissue of the renal capsule, and consists of two to three layers of irregularly shaped mesenchymal cells that stain deeply with toluidine blue. The nephrons are in different stages of maturation; those nearest the renal pelvis are more differentiated than those in the peripheral cortex. Near the capsule, the nephrons are very immature in appearance and are in the initial stages of differentiation (Figs. 2-6). Although the nephron units in the metanephros of the newborn opossum vary considerably in their degree of maturation, all appear to follow the same pattern of glomerular and tubular differentiation and development. In the initial stages of development the expanded branches of the collecting tubules (ampullae) that lie immediately beneath
Fig. 1. A scanning electron micrograph of the posterior abdominal wall showing the adrenal cortex (A), metanephros (mt), mesonephros (ms), gonad (g), colon (c), mesonephric (md) and Mullerian ducts (mu). Newborn opossum. x 50. Fig. 2. The newborn metanephros cut near the renal pelvis shows numerous collecting tubules (ct), occasional glomeruli (g) and differentiating nephrons (arrows). Haematoxylin and eosin. x 100. Fig. 3. The cortical region adjacent to the capsule shows the ampulla of a collecting tubule (large arrow) surrounded by a mass of nephronogenic cells. Note the mitotic activity within this group of cells. A portion of collecting tubule (ct) and the tubular components of an adjacent nephron also are shown. Newborn opossum. Epon 812. Toluidine blue. x 400. Fig. 4. A developing nephron from a newborn opossum. A portion of a collecting tubule (ct) is shown near the top of the figure. The region of the forming renal corpuscle shows flattened peripheral cells (small arrows) which form the parietal layer of Bowman's capsule, and an adjacent layer of cuboidal cells (the forming visceral layer). A capillary (large arrows) is shown entering the developing corpuscle at the left, adjacent to the tubular portion of the nephron. The capillary expands near the centre immediately subjacent to the surrounding visceral layer of Bowman's capsule. Epon 812. Toluidine blue. x 400. Fig. 5. A developing nephron showing a renal corpuscle that is more differentiated than the one depicted in Fig. 4. The capsular space is established and the visceral layer (arrows) continues to differentiate in relation to the forming glomerulus. Newborn opossum. Epon 812. Toluidine blue. x 400. Fig. 6. A renal corpuscle showing further differentiation than that illustrated in Figs. 4 and 5. The parietal layer, visceral layer, glomerular capillary loops, capsular space and urinary pole (arrow) are clearly shown. Newborn opossum. Epon 812. Toluidine blue. x 400.
462
W. J. KRAUSE, J. H. CUTTS AND C. R. LEESON
APOW... C, A
B
D
7
Fig. 7. A schematic representation of early nephronogenesis and the formation of a renal corpuscle in the opossum metanephros. Initially, branches of the dividing collecting tubule (ampullae) are surrounded by primitive mesenchymal cells that migrate from a nephronogenic zone located immediately beneath the renal capsule (A). Some of these cells form a thin doublelayered cap around the expanding branches of the collecting tubule as a loose, oval aggregate of cells forms between the collecting tubule and its branch (A, B). As a central cavity appears, a single layer of component cells is arranged with the long axis of its cells perpendicular to the lumen. The formed renal vesicle shows considerable mitotic activity as it elongates (C). Two indentations then appear and transform the elongate form into a blind-ended, S-shaped tubule (D). The lower limb of the tubule (that furthermost from the cortex or the branch of the collecting tubule) will form the renal corpuscle; the middle limb will become the proximal tubule; the upper limb will form the loop of Henle and the distal tubule. As the S-shaped form is established, [Continued on factng page
The metanephros of the postnatal opossum
463
the renal capsule become surrounded by nephronogenic cells (Figs. 3, 7), which coalesce to form a solid oval mass in relation to the ampulla and to the collecting tubule. A central cavity then appears and a single layer of columnar cells forms, with the long axes of the cells lying perpendicular to the lumen. Continued division of the cells of the newly formed renal vesicle results in the formation of an elongated tubule. As the tubule continues to elongate, two indentations occur and transform it into an S-shaped tubule (Figs. 2, 7). The lower limb of the S-shaped structure, which is furthest from the external surface of the kidney, will differentiate into the renal corpuscle. The cells of the lower limb begin to flatten, whereas epithelial cells on the opposite side of the same limb appear to enlarge slightly (Fig. 7). Simultaneously a small capillary penetrates the area between the lower and middle limbs of the S-shaped tubule to provide the vascular component of the forming renal corpuscle. The developing capillaries become intimately associated with the columnar epithelial cells that are continuous with those of the remainder of the tubule (Figs. 4-7). As the visceral layer of Bowman's capsule differentiates and develops around the invading vasculature, the cells on the opposite side of this portion of the tubule form the simple squamous epithelium of the parietal layer of Bowman's capsule (Fig. 7). The lumen at this end of the S-shaped tubule is the capsular space of the forming renal corpuscle. As the glomerular capillaries become established the parietal layer of Bowman's capsule appears to constrict around the original vascular point of entry to establish the vascular pole. The capsular space of the forming renal corpuscle remains continuous with the lumen of the remainder of the nephron unit, and forms the urinary pole (Figs. 6,7). Considerable mitotic activity is observed in the expanding proximal and distal limbs of the tubular component of the developing nephron. Communication between the developing nephron unit and the collecting tubule is established concomitant with the differentiation of the renal corpuscle (Fig. 7). Ultrastructurally, cells of the nephronogenic layer and of the aggregate near the expanding branch of the collecting tubule appear irregular in shape and are without any apparent organization. The cytoplasm is of considerable electron density and contains numerous polyribosomes (Fig. 8). Occasional profiles of granular endoplasmic reticulum and small, scattered mitochondria also are observed. Nuclei are irregular and contain prominent nucleoli. After the parietal and visceral layers of Bowman's capsule have been established, the podocytes which make up the visceral layer show a definite orientation along the developing capillaries (Figs. 4-6, 9, 10). The differentiating podocytes appear dome-like and uniform in shape (Figs. 5, 6, 9, 10), and that surface which is associated with the developing glomerular capillaries lies on a delicate basal lamina (Figs. 9, 10). The podocytes often show long microprojections extending from their surface, and occasional cilia are observed (Fig. 10). Cells of the visceral layer show an electrondense cytoplasm and numerous free polyribosomes. When compared to the cells of a capillary enters the space between the middle and lower limbs of the tubule (E) to become intimately associated with the epithelium of this limb (E, F). The tubular epithelium associated with the forming capillaries remains cuboidal to columnar, whereas cells composing the remainder of this limb flatten to form the parietal layer of Bowman's capsule (F, G, H). As these events occur the forming nephron joins the collecting tubule (E, F). The parietal layer expands and appears to constrict around the point of entry of the capillary, resulting in the formation of the vascular pole (G, H). The remaining tubular epithelium covering the forming capillaries undergoes marked morphological changes and forms the visceral (podocytic) layer of Bowman's capsule (G, H).
464
W. J. KRAUSE, J. H. CUTTS AND C. R. LEESON ._ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~AL
Cs WeZK
a
Zfe ~~~~~~~~~~~~P
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