Cell and Tissue Research
Cell Tiss. Res. 182, 557-564 (1977)
9 by Springer-Verlag 1977
The Ultrastructure of the Sinus Gland of Gammarus oceanicus (Crustacea: Amphipoda) D.A. Brodie and K. Halcrow* Biology Department, University of New Brunswick, Saint John, New Brunswick, Canada
Summary. The sinus gland of
G a m m a r u s oceanieus, like that of other crustaceans, is composed of three elements: neurosecretory axons, glial cells and stromal sheath. Five neurosecretory axon types are identified on the basis of granule diameter, shape, and electron density, and axon matrix density. Exocytosis appears to be the major release mechanism of neurosecretory material. The preterminal regions of neurosecretory axons contain axoplasmic reticulum and neurotubules. Their arrangement in the axon and relationship with one another suggest a transport function. Multilamellar bodies are found in the terminal regions of neurosecretory axons. They arise from mitochondria and may be involved in granulolysis.
Key words: Sinus gland -
Gammarus -
Neurosecretion - Ultrastructure.
Introduction The crustacean sinus gland is a neurohemal organ involved in the storage and release of neurosecretory material subserving several endocrine functions (see Gabe, 1966; Kleinholz, 1976; Highnam and Hill, 1977). There is much interest in the identification of types ofneurosecretory material and their release mechanisms. The neurosecretory products appear as granules; their diameter, shape and electron density, and the density of the axon matrix have been used to characterize various types (Hodge and Chapman, 1958; Fingerman and Aoto, 1959; Knowles, 1959; Bunt and Ashby, 1967; Meusy, 1968; Shivers, 1969; Weitzman, 1969; Andrews et al., 1971; Martin, 1972; Smith, 1974; Silverthorn, 1975; Bressac, 1976). Only two of these investigations deal with non-decapod malacostracan sinus glands (Knowles, Send offprint requests to: Dr. D.A. Brodie, Biology Department, U.N.B.S.J., P.O. Box 5050, Saint John, N.B., E2L 4L5, Canada
* The technical assistance of G.A. Bance, statistical assistance of D. MacCharles and D.W. Hagen, and financial support provided by the University of New Brunswick Research Fund to K.H. are gratefully acknowledged
558
D.A. Brodie and K. Halcrow
1959; M a r t i n , 1972). T h e p r e s e n t p a p e r d e s c r i b e s the u l t r a s t r u c t u r e o f the sinus g l a n d o f a g a m m a r i d , G a m m a r u s oeeanicus, w h i c h r e p r e s e n t s a m a l a c o s t r a c a n o r d e r ( A m p h i p o d a ) n o t p r e v i o u s l y s t u d i e d in this respect.
Materials and Methods Male Gammarus oceanicus, 17-22 mm in length and in stage C of the molt cycle (Charniaux-Legrand, 1952), were decapitated and the heads fixed in ice-cold phosphate-buffered 2.5% glutaraldehyde containing 0.28 M sucrose at pH 7.2 for 2 to 61/2 h. The eye regions were then placed in fresh fixative for 30 min. Total fixation time varied from 31/2 to 8 h. Specimens were then rinsed in a buffer wash for 5 min. Postfixation was carried out with ice-cold phosphate-buffered 2.0% osmium tetroxide containing 0.65 M sucrose at pH 7.2 for 30min. Specimens were again rinsed in buffer wash for 5min. The osmolality and pH of the fixatives were adjusted according to Maser et al. (1967) and confirmed by osmometer and pH meter readings. Tissue embedded in the hard formula of Spurr's resin (Spurr, 1969), sectioned and stained with uranyl acetate and lead citrate, was examined in Philips EM 75 and Philips EM 200 electron microscopes. Paraffin sections stained with aldehyde-fuchsin (Ewen, 1962) and plastic sections stained with 1% toluidine blue in 1% sodium tetraborate were examined by light microscopy. Granule diameter was determined by randomly selecting no more than 20 axon profiles (computer limit) per sinus gland and measuring the diameter of 25 granules chosen at random from each axon. These granules are ovoid to round in section and each possesses a smooth limiting membrane. Scattered among these granules used in axon classification are granules bounded by wavy membranes, with a "haloed" appearance and of low electron density. Tailed and dumb-bell shaped granules are also found and may represent two or more granules close to one another and sectioned at such an angle as to appear as one. These granules were not used in axon classification. Measurements were taken from prints viewed through a dissecting microscope such that granules were observed at a total magnification of 81,300 • Twelve sinus glands and a total of 198 axon endings were surveyed in this manner. One way analysis of variance followed by a posteriori tests (SNK and Scheffe's) were used on mean granule diameter to determine boundaries for each axon type (Zar, 1974). Axons were further classified according to granule shape and electron density, and axon matrix density with frequency histograms.
Results T h r o u g h t h e light m i c r o s c o p e , t h e sinus g l a n d a p p e a r s as a c o l l e c t i o n o f a x o n s a n d h e a v i l y - s t a i n e d e n d i n g s p o s t e r o - v e n t r a l to the o p t i c lobe. T h i s is in a c c o r d a n c e w i t h G r / i b e r (1933) w h o s e " p s e u d o f r o n t a l o r g a n " r e p r e s e n t s the sinus gland. Its s t r o m a l s h e a t h is c o n t i n u o u s w i t h the n e u r o l e m m a o f t h e o p t i c l o b e as s h o w n b y St&hl (1938), T h e p o i n t o f e m e r g e n c e o f the n e u r o s e c r e t o r y a x o n s f r o m the l a m i n a g a n g l i o n a r i s is t a k e n as the b o u n d a r y b e t w e e n o p t i c l o b e a n d sinus gland. T h e l a t t e r is 2 5 0 - 3 0 0 lam in l e n g t h a n d 4 0 - 5 0 lam in d i a m e t e r at its w i d e s t point. T h e e l e c t r o n m i c r o s c o p e reveals t h r e e m a j o r e l e m e n t s : n e u r o s e c r e t o r y axons, glial cells a n d s t r o m a l sheath. T h e s t r o m a l s h e a t h f o r m s a n a c e l l u l a r b o u n d a r y b e t w e e n the a x o n s a n d h e m o l y m p h a n d varies f r o m 1 5 0 - 5 0 0 n m in thickness. T h e s u r f a c e o f the sinus g l a n d is c o n v o l u t e d a n d e x t e n s i o n s o f t h e s t r o m a l s h e a t h r e a c h t h e interior. T h e p r e t e r m i n a l r e g i o n s o f the n e u r o s e c r e t o r y a x o n s o c c u p y m u c h o f the p r o x i m a l a n d c e n t r a l p o r t i o n s o f the g l a n d a n d are c h a r a c t e r i z e d b y n e u r o t u b u l e s a n d a x o p l a s m i c r e t i c u l u m (Fig. 1). T h e a x o p l a s m i c r e t i c u l u m consists o f a series o f
Fig. 1. Axoplasmic reticulum in preterminal region of neurosecretory axon. Note transverse (t), longitudinal (/) and vesicular (v) elements, neurotubule (nt) passing through gap in axoplasmic reticulum. x 14,900 Fig. 2. Axoplasmic reticulum (at) continuous with axolemma. • 13,400 Figs. 3-5. Axon types of sinus gland o f G a m m a r u s oceanicus. I type 1 ; 2 type 2; 3 type 3; 4 type 4; 5 type 5; m mitochondrion, x 13,400 Fig. 6. Exocytotic figure containing neurosecretory material (arrow). Note granules bounded by wavy membrane and with weakly stained contents (wg). * stromal sheath. • 38,400 Fig. 7. Neurosecretory granule attached to axolemma by connecting membrane (arrow). • 38,400
TaMe 1. Neurosecretory axon types in the sinus gland of Gammarus oceanicus Axon type
Mean granule diameter range (nm)
Granule diameter range (nm)
Granule electron density
Granule shape
Axon matrix density
1
70-110 105-155 90-150 100-155 155-200
60-140 70-190 70-210 70-200 90-315
dense dense very dense not dense not dense
irregular to oval/round oval/round oval/round oval/round oval/round
not dense dense not dense not dense not dense
2 3 4 5
|
| o x
0 x
Cell Tiss. Res. 182, 557-564 (1977)
9 by Springer-Verlag 1977
The Ultrastructure of the Sinus Gland of Gammarus oceanicus (Crustacea: Amphipoda) D.A. Brodie and K. Halcrow* Biology Department, University of New Brunswick, Saint John, New Brunswick, Canada
Summary. The sinus gland of
G a m m a r u s oceanieus, like that of other crustaceans, is composed of three elements: neurosecretory axons, glial cells and stromal sheath. Five neurosecretory axon types are identified on the basis of granule diameter, shape, and electron density, and axon matrix density. Exocytosis appears to be the major release mechanism of neurosecretory material. The preterminal regions of neurosecretory axons contain axoplasmic reticulum and neurotubules. Their arrangement in the axon and relationship with one another suggest a transport function. Multilamellar bodies are found in the terminal regions of neurosecretory axons. They arise from mitochondria and may be involved in granulolysis.
Key words: Sinus gland -
Gammarus -
Neurosecretion - Ultrastructure.
Introduction The crustacean sinus gland is a neurohemal organ involved in the storage and release of neurosecretory material subserving several endocrine functions (see Gabe, 1966; Kleinholz, 1976; Highnam and Hill, 1977). There is much interest in the identification of types ofneurosecretory material and their release mechanisms. The neurosecretory products appear as granules; their diameter, shape and electron density, and the density of the axon matrix have been used to characterize various types (Hodge and Chapman, 1958; Fingerman and Aoto, 1959; Knowles, 1959; Bunt and Ashby, 1967; Meusy, 1968; Shivers, 1969; Weitzman, 1969; Andrews et al., 1971; Martin, 1972; Smith, 1974; Silverthorn, 1975; Bressac, 1976). Only two of these investigations deal with non-decapod malacostracan sinus glands (Knowles, Send offprint requests to: Dr. D.A. Brodie, Biology Department, U.N.B.S.J., P.O. Box 5050, Saint John, N.B., E2L 4L5, Canada
* The technical assistance of G.A. Bance, statistical assistance of D. MacCharles and D.W. Hagen, and financial support provided by the University of New Brunswick Research Fund to K.H. are gratefully acknowledged
558
D.A. Brodie and K. Halcrow
1959; M a r t i n , 1972). T h e p r e s e n t p a p e r d e s c r i b e s the u l t r a s t r u c t u r e o f the sinus g l a n d o f a g a m m a r i d , G a m m a r u s oeeanicus, w h i c h r e p r e s e n t s a m a l a c o s t r a c a n o r d e r ( A m p h i p o d a ) n o t p r e v i o u s l y s t u d i e d in this respect.
Materials and Methods Male Gammarus oceanicus, 17-22 mm in length and in stage C of the molt cycle (Charniaux-Legrand, 1952), were decapitated and the heads fixed in ice-cold phosphate-buffered 2.5% glutaraldehyde containing 0.28 M sucrose at pH 7.2 for 2 to 61/2 h. The eye regions were then placed in fresh fixative for 30 min. Total fixation time varied from 31/2 to 8 h. Specimens were then rinsed in a buffer wash for 5 min. Postfixation was carried out with ice-cold phosphate-buffered 2.0% osmium tetroxide containing 0.65 M sucrose at pH 7.2 for 30min. Specimens were again rinsed in buffer wash for 5min. The osmolality and pH of the fixatives were adjusted according to Maser et al. (1967) and confirmed by osmometer and pH meter readings. Tissue embedded in the hard formula of Spurr's resin (Spurr, 1969), sectioned and stained with uranyl acetate and lead citrate, was examined in Philips EM 75 and Philips EM 200 electron microscopes. Paraffin sections stained with aldehyde-fuchsin (Ewen, 1962) and plastic sections stained with 1% toluidine blue in 1% sodium tetraborate were examined by light microscopy. Granule diameter was determined by randomly selecting no more than 20 axon profiles (computer limit) per sinus gland and measuring the diameter of 25 granules chosen at random from each axon. These granules are ovoid to round in section and each possesses a smooth limiting membrane. Scattered among these granules used in axon classification are granules bounded by wavy membranes, with a "haloed" appearance and of low electron density. Tailed and dumb-bell shaped granules are also found and may represent two or more granules close to one another and sectioned at such an angle as to appear as one. These granules were not used in axon classification. Measurements were taken from prints viewed through a dissecting microscope such that granules were observed at a total magnification of 81,300 • Twelve sinus glands and a total of 198 axon endings were surveyed in this manner. One way analysis of variance followed by a posteriori tests (SNK and Scheffe's) were used on mean granule diameter to determine boundaries for each axon type (Zar, 1974). Axons were further classified according to granule shape and electron density, and axon matrix density with frequency histograms.
Results T h r o u g h t h e light m i c r o s c o p e , t h e sinus g l a n d a p p e a r s as a c o l l e c t i o n o f a x o n s a n d h e a v i l y - s t a i n e d e n d i n g s p o s t e r o - v e n t r a l to the o p t i c lobe. T h i s is in a c c o r d a n c e w i t h G r / i b e r (1933) w h o s e " p s e u d o f r o n t a l o r g a n " r e p r e s e n t s the sinus gland. Its s t r o m a l s h e a t h is c o n t i n u o u s w i t h the n e u r o l e m m a o f t h e o p t i c l o b e as s h o w n b y St&hl (1938), T h e p o i n t o f e m e r g e n c e o f the n e u r o s e c r e t o r y a x o n s f r o m the l a m i n a g a n g l i o n a r i s is t a k e n as the b o u n d a r y b e t w e e n o p t i c l o b e a n d sinus gland. T h e l a t t e r is 2 5 0 - 3 0 0 lam in l e n g t h a n d 4 0 - 5 0 lam in d i a m e t e r at its w i d e s t point. T h e e l e c t r o n m i c r o s c o p e reveals t h r e e m a j o r e l e m e n t s : n e u r o s e c r e t o r y axons, glial cells a n d s t r o m a l sheath. T h e s t r o m a l s h e a t h f o r m s a n a c e l l u l a r b o u n d a r y b e t w e e n the a x o n s a n d h e m o l y m p h a n d varies f r o m 1 5 0 - 5 0 0 n m in thickness. T h e s u r f a c e o f the sinus g l a n d is c o n v o l u t e d a n d e x t e n s i o n s o f t h e s t r o m a l s h e a t h r e a c h t h e interior. T h e p r e t e r m i n a l r e g i o n s o f the n e u r o s e c r e t o r y a x o n s o c c u p y m u c h o f the p r o x i m a l a n d c e n t r a l p o r t i o n s o f the g l a n d a n d are c h a r a c t e r i z e d b y n e u r o t u b u l e s a n d a x o p l a s m i c r e t i c u l u m (Fig. 1). T h e a x o p l a s m i c r e t i c u l u m consists o f a series o f
Fig. 1. Axoplasmic reticulum in preterminal region of neurosecretory axon. Note transverse (t), longitudinal (/) and vesicular (v) elements, neurotubule (nt) passing through gap in axoplasmic reticulum. x 14,900 Fig. 2. Axoplasmic reticulum (at) continuous with axolemma. • 13,400 Figs. 3-5. Axon types of sinus gland o f G a m m a r u s oceanicus. I type 1 ; 2 type 2; 3 type 3; 4 type 4; 5 type 5; m mitochondrion, x 13,400 Fig. 6. Exocytotic figure containing neurosecretory material (arrow). Note granules bounded by wavy membrane and with weakly stained contents (wg). * stromal sheath. • 38,400 Fig. 7. Neurosecretory granule attached to axolemma by connecting membrane (arrow). • 38,400
TaMe 1. Neurosecretory axon types in the sinus gland of Gammarus oceanicus Axon type
Mean granule diameter range (nm)
Granule diameter range (nm)
Granule electron density
Granule shape
Axon matrix density
1
70-110 105-155 90-150 100-155 155-200
60-140 70-190 70-210 70-200 90-315
dense dense very dense not dense not dense
irregular to oval/round oval/round oval/round oval/round oval/round
not dense dense not dense not dense not dense
2 3 4 5
|
| o x
0 x
