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The Effect of Inherited Chondrodystrophy on the Hexosamine Content of Cartilage from Turkey Embryos
The Effect of Inherited Chondrodystrophy on the Hexosamine Content of Cartilage from Turkey Embryos R. M. LEACH, JR. and E. G. BUSS Department of Poultry Science, Pennsylvania State University, University Park, Pennsylvania 16802 (Received for publication November 16, 1976)
Poultry Science 56:1043-1045, 1977 INTRODUCTION Manganese deficiency in chickens results in severe impairment of skeletal development. A deficiency during embryonic development leads to chondrodystrophy (Lyons and Insko, 1937) while in the young chick the skeletal deformity has been termed perosis (Wilgus et al., 1936). An impairment in mucopolysaccharide biosynthesis appears to be the biochemical explanation for these lesions (Leach and Muenster, 1962). In addition to nutritionally induced skeletal lesions, a number of inherited forms of embryonic skeletal deformities have been reported (Landauer, 1967). Nanomelia, one of the deformities observed in chickens, has been shown to be associated with changes in cartilage mucopolysaccharide content (Mathews, 1967). Recently, an inherited embryonic chondrodystrophy has been described in turkeys (Gaffney, 1975). The purpose of this investigation was to characterize the chemical and histological changes which are associated with chondrodystrophy in the embryonic turkey poult. MATERIALS AND METHODS Embryos were sacrificed at 21 days of incubation. The cartilaginous ends of the long bones (femur, tibiotarsus and tarsalmetatarsus)
1 Decal, Omega Chemical Corp., Cold Spring, New York 10516. 2 Armed Forces Institute of Pathology, 1960. Manual of Histologic and Special Staining Techniques, Ed. 2. McGraw-Hill Book Company, New York, pp. 142-143.
were removed, frozen and subsequently lyophilized. The dry cartilage was fat extracted with chloroform-methanol and ground into a fine powder. Hexosamine analysis was used to estimate the mucopolysaccharide content of the cartilage. The lipid-free dry cartilage was hydrolyzed in sealed tubes according to the procedure of Anastassiadis and Common (1958). Total hexosamine was determined by the method of Boas (1953). A modification of the column chromatographic procedure of Gardell (1953) was used to quantitate the amount of glucosamine and galactosamine. Tibiotarsi were used for histological examination. The bone was removed, cleaned of adhering tissue, fixed in 10 percent buffered formalin and decalcified in Decal . Parafin imbedded sections were stained with Alcian Blue - PAS 2 . RESULTS AND DISCUSSION The results of the hexosamine analysis are presented in Table 1. The values shown represent the average of duplicate analyses on three pooled samples obtained from three different hatches during two hatching seasons. The cartilage from six or more embryos was pooled to provide each sample. Cartilage from embryos with chondrodystrophy contained less than one-half the amount of hexosamine found in normal embryonic cartilage. A decrease in the galactosamine containing mucopolysaccharides was responsible for this reduction. Histological examination of representative embryonic limbs confirmed the effect of chondrodystrophy on cartilage extracellular matrix.
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Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 4, 2015
ABSTRACT The effect of inherited chondrodystrophy on the composition of embryonic turkey cartilage was studied. Cartilage from embryos homozygous for the mutant gene contained less than one-half the normal amount of galactosamine containing mucopolysaccharides. Histological examination also showed that there was a substantial decrease in extracellular matrix content of the chondrodystrophic cartilage.
LEACH AND BUSS
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TABLE 1. — Effect of chondrodystrophy
on the composition of embryonic cartilage
Epiphyseal cartilage hexosamine content
Type of embryo
Glucosamine
Galactosamine
mg./gm.1
mg./gm.
mg./gm.
2.09 ± 0.41 2 0.97 t 0.39 a
0.29 ± 0.07 0.39±O.12 b
1.80 ±0.28 0.58 ± 0.30 a
Total
Normal ch/ch 1
Fat-free dry cartilage.
2
Mean ± standard deviation. Significantly different from normal p<0.05 based upon analysis of variance.
In Figure 2 is presented t h e typical appearance of a section of a tibiotarsus of a c h o n d r o d y s t r o p h i c limb. Comparison with t h e normal b o n e , Figure 1, indicates t h a t there is a substantial r e d u c t i o n in t h e extracellular matrix surr o u n d i n g t h e c h o n d r o c y t e s in t h e c h o n d r o d y s t r o p h i c e m b r y o n i c limb. These changes in c o m p o s i t i o n and histology are very similar t o t h o s e observed with manganese deficient chicks r e p o r t e d by Leach and Muenster ( 1 9 6 2 ) , and Leach ( 1 9 6 8 ) . It is also a p p a r e n t t h a t these gross changes in t h e c h o n d r o d y s t r o p h i c t u r k e y e m b r y o s are very similar t o t h e changes observed b y Mathews
( 1 9 6 7 ) for t h e limbs of nanomelic chick embryos. Fraser and Goetinck ( 1 9 7 1 ) used radioactive substrates to show that c h o n d r o i t i n sulfate synthesis was reduced in nanomelic sternal cartilage. F u r t h e r studies were conducted by Palmoski and Goetinck ( 1 9 7 2 ) using nanomelic c h o n d r o c y t e s grown in tissue cult u r e . Isolation and fractionation of t h e chondroitin sulfate synthesized by these cells showed t h a t t h e major c h r o m a t o g r a p h i c comp o n e n t was absent in these m u t a n t c h o n d r o cytes. At t h e present t i m e it is n o t k n o w n if a similar p a t t e r n exists for t h e c h o n d r o d y s t r o p h i c turkey embryo.
mn tm 4PI. FIG. 1. Chondrocytes in the embryonic cartilage cone of a normal 21-day turkey embryo. 250X.
FIG. 2. Chondrocytes in the embryonic cartilage cone of a chondrodystrophic 21-day turkey embryo. 250X.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 4, 2015
Significantly different from normal p<0.01 based upon analysis of variance.
RESEARCH NOTES ACKNOWLEDGEMENT T h e a u t h o r s wish t o a c k n o w l e d g e t h e technical assistance of Mrs. Bonnie Seely and Mr. L. F. Gaffney in t h e collection of analysis of t h e e m b r y o n i c t u r k e y cartilage.
REFERENCES
Scand. 7:207-215. Landauer, W., 1967. The hatchability of chicken eggs as influenced by environment and heredity: Monograph I (Revised), Storrs Agricultural Experiment Sta. pp. 1 4 3 - 1 8 5 . Leach, R. M., Jr., 1968. Effect of manganese upon the epiphyseal growth plate in the young chick. Poultry Sci. 4 7 : 8 2 8 - 8 3 0 . Leach, R. M., Jr., and A. M. Muenster, 1962. Studies on the role of manganese in bone formation. I. Effect upon mucopolysaccharide content of chick bone. J. Nutr. 78:51-56. Lyons, M., and W. M. Insko, Jr., 1937. Chondrodystrophy in the chick embryo produced by manganese deficiency in the diet of the hen. Kentucky Agric. Sta. Bull. No. 371:63-75. Mathews, M. B., 1967. Chondroitin sulfate and colagen in inherited skeletal defects of chickens. Nature, 213:1255-1256. Palmoski, M. J., and P. F. Goetinck, 1972. Synthesis of proteochondroitin sulfate by normal, nanomelic, and 5-bromodeoxyuridine-treated chondrocytes in cell culture. Proc. Nat. Acad. Sci. 69:3385-3388. Wilgus, H. S., L. C. Norris and G. F. Heuser, 1936. The role of certain inorganic elements in the cause and prevention of perosis. Science, 84:252—253.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 4, 2015
Anastassiadis, P. A., and R. H. Common, 1958. Liberation of hexosamine, hexuronic acid and hydroxyproiine from tissue dry resin hydrolysis. Canad. J. Biochem. Physiol. 36:413-424. Boas, N. F., 1953. Method for the determination of h e x o s a m i n e in t i s s u e s . J. Biol. Chem. 204:553-563. Fraser, R. A., and P. F. Goetinck, 1971. Reduced synthesis of chondroitin sulfate by cartilage from the mutant, nanomelia. Biochem. Biophys. Res. Commun. 4 3 : 4 9 4 - 5 0 3 . Gaffney, L. J., 1975. Chondrodystrophy: an inherited lethal condition in turkey embryos. J. Heredity, 66:339-343. Gardell, S., 1953. Separation on Dowex 50 ion-exchange resin of glucosamine and galactosamine, and their quantitative determination. Acta Chem.
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Poultry Science 56:1043-1045, 1977 INTRODUCTION Manganese deficiency in chickens results in severe impairment of skeletal development. A deficiency during embryonic development leads to chondrodystrophy (Lyons and Insko, 1937) while in the young chick the skeletal deformity has been termed perosis (Wilgus et al., 1936). An impairment in mucopolysaccharide biosynthesis appears to be the biochemical explanation for these lesions (Leach and Muenster, 1962). In addition to nutritionally induced skeletal lesions, a number of inherited forms of embryonic skeletal deformities have been reported (Landauer, 1967). Nanomelia, one of the deformities observed in chickens, has been shown to be associated with changes in cartilage mucopolysaccharide content (Mathews, 1967). Recently, an inherited embryonic chondrodystrophy has been described in turkeys (Gaffney, 1975). The purpose of this investigation was to characterize the chemical and histological changes which are associated with chondrodystrophy in the embryonic turkey poult. MATERIALS AND METHODS Embryos were sacrificed at 21 days of incubation. The cartilaginous ends of the long bones (femur, tibiotarsus and tarsalmetatarsus)
1 Decal, Omega Chemical Corp., Cold Spring, New York 10516. 2 Armed Forces Institute of Pathology, 1960. Manual of Histologic and Special Staining Techniques, Ed. 2. McGraw-Hill Book Company, New York, pp. 142-143.
were removed, frozen and subsequently lyophilized. The dry cartilage was fat extracted with chloroform-methanol and ground into a fine powder. Hexosamine analysis was used to estimate the mucopolysaccharide content of the cartilage. The lipid-free dry cartilage was hydrolyzed in sealed tubes according to the procedure of Anastassiadis and Common (1958). Total hexosamine was determined by the method of Boas (1953). A modification of the column chromatographic procedure of Gardell (1953) was used to quantitate the amount of glucosamine and galactosamine. Tibiotarsi were used for histological examination. The bone was removed, cleaned of adhering tissue, fixed in 10 percent buffered formalin and decalcified in Decal . Parafin imbedded sections were stained with Alcian Blue - PAS 2 . RESULTS AND DISCUSSION The results of the hexosamine analysis are presented in Table 1. The values shown represent the average of duplicate analyses on three pooled samples obtained from three different hatches during two hatching seasons. The cartilage from six or more embryos was pooled to provide each sample. Cartilage from embryos with chondrodystrophy contained less than one-half the amount of hexosamine found in normal embryonic cartilage. A decrease in the galactosamine containing mucopolysaccharides was responsible for this reduction. Histological examination of representative embryonic limbs confirmed the effect of chondrodystrophy on cartilage extracellular matrix.
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Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 4, 2015
ABSTRACT The effect of inherited chondrodystrophy on the composition of embryonic turkey cartilage was studied. Cartilage from embryos homozygous for the mutant gene contained less than one-half the normal amount of galactosamine containing mucopolysaccharides. Histological examination also showed that there was a substantial decrease in extracellular matrix content of the chondrodystrophic cartilage.
LEACH AND BUSS
1044
TABLE 1. — Effect of chondrodystrophy
on the composition of embryonic cartilage
Epiphyseal cartilage hexosamine content
Type of embryo
Glucosamine
Galactosamine
mg./gm.1
mg./gm.
mg./gm.
2.09 ± 0.41 2 0.97 t 0.39 a
0.29 ± 0.07 0.39±O.12 b
1.80 ±0.28 0.58 ± 0.30 a
Total
Normal ch/ch 1
Fat-free dry cartilage.
2
Mean ± standard deviation. Significantly different from normal p<0.05 based upon analysis of variance.
In Figure 2 is presented t h e typical appearance of a section of a tibiotarsus of a c h o n d r o d y s t r o p h i c limb. Comparison with t h e normal b o n e , Figure 1, indicates t h a t there is a substantial r e d u c t i o n in t h e extracellular matrix surr o u n d i n g t h e c h o n d r o c y t e s in t h e c h o n d r o d y s t r o p h i c e m b r y o n i c limb. These changes in c o m p o s i t i o n and histology are very similar t o t h o s e observed with manganese deficient chicks r e p o r t e d by Leach and Muenster ( 1 9 6 2 ) , and Leach ( 1 9 6 8 ) . It is also a p p a r e n t t h a t these gross changes in t h e c h o n d r o d y s t r o p h i c t u r k e y e m b r y o s are very similar t o t h e changes observed b y Mathews
( 1 9 6 7 ) for t h e limbs of nanomelic chick embryos. Fraser and Goetinck ( 1 9 7 1 ) used radioactive substrates to show that c h o n d r o i t i n sulfate synthesis was reduced in nanomelic sternal cartilage. F u r t h e r studies were conducted by Palmoski and Goetinck ( 1 9 7 2 ) using nanomelic c h o n d r o c y t e s grown in tissue cult u r e . Isolation and fractionation of t h e chondroitin sulfate synthesized by these cells showed t h a t t h e major c h r o m a t o g r a p h i c comp o n e n t was absent in these m u t a n t c h o n d r o cytes. At t h e present t i m e it is n o t k n o w n if a similar p a t t e r n exists for t h e c h o n d r o d y s t r o p h i c turkey embryo.
mn tm 4PI. FIG. 1. Chondrocytes in the embryonic cartilage cone of a normal 21-day turkey embryo. 250X.
FIG. 2. Chondrocytes in the embryonic cartilage cone of a chondrodystrophic 21-day turkey embryo. 250X.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 4, 2015
Significantly different from normal p<0.01 based upon analysis of variance.
RESEARCH NOTES ACKNOWLEDGEMENT T h e a u t h o r s wish t o a c k n o w l e d g e t h e technical assistance of Mrs. Bonnie Seely and Mr. L. F. Gaffney in t h e collection of analysis of t h e e m b r y o n i c t u r k e y cartilage.
REFERENCES
Scand. 7:207-215. Landauer, W., 1967. The hatchability of chicken eggs as influenced by environment and heredity: Monograph I (Revised), Storrs Agricultural Experiment Sta. pp. 1 4 3 - 1 8 5 . Leach, R. M., Jr., 1968. Effect of manganese upon the epiphyseal growth plate in the young chick. Poultry Sci. 4 7 : 8 2 8 - 8 3 0 . Leach, R. M., Jr., and A. M. Muenster, 1962. Studies on the role of manganese in bone formation. I. Effect upon mucopolysaccharide content of chick bone. J. Nutr. 78:51-56. Lyons, M., and W. M. Insko, Jr., 1937. Chondrodystrophy in the chick embryo produced by manganese deficiency in the diet of the hen. Kentucky Agric. Sta. Bull. No. 371:63-75. Mathews, M. B., 1967. Chondroitin sulfate and colagen in inherited skeletal defects of chickens. Nature, 213:1255-1256. Palmoski, M. J., and P. F. Goetinck, 1972. Synthesis of proteochondroitin sulfate by normal, nanomelic, and 5-bromodeoxyuridine-treated chondrocytes in cell culture. Proc. Nat. Acad. Sci. 69:3385-3388. Wilgus, H. S., L. C. Norris and G. F. Heuser, 1936. The role of certain inorganic elements in the cause and prevention of perosis. Science, 84:252—253.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 4, 2015
Anastassiadis, P. A., and R. H. Common, 1958. Liberation of hexosamine, hexuronic acid and hydroxyproiine from tissue dry resin hydrolysis. Canad. J. Biochem. Physiol. 36:413-424. Boas, N. F., 1953. Method for the determination of h e x o s a m i n e in t i s s u e s . J. Biol. Chem. 204:553-563. Fraser, R. A., and P. F. Goetinck, 1971. Reduced synthesis of chondroitin sulfate by cartilage from the mutant, nanomelia. Biochem. Biophys. Res. Commun. 4 3 : 4 9 4 - 5 0 3 . Gaffney, L. J., 1975. Chondrodystrophy: an inherited lethal condition in turkey embryos. J. Heredity, 66:339-343. Gardell, S., 1953. Separation on Dowex 50 ion-exchange resin of glucosamine and galactosamine, and their quantitative determination. Acta Chem.
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