N e u r o c h e m i c a l R e s e a r c h (2) 11-25 (1977)
CHANGES IN BRAIN COMPONENTS D U R I N G THE D E V E L O P M E N T OF MICE H O M O Z Y G O U S FOR THE L O C U S " D W A R F " (dw) 1'5 A. LEES,2 L. L. SARLII~VE, 3 N. M. NESKOVIC,3 M.
WINTZERITH,
4 AND P.
MANDEL
Centre de Neurochimie du CNRS and Institut de Chimie Biologique FacultO de M~decine I1, Rue Humann, 67085 Strasbourg Cedex, France
Accepted August 13, 1976
Brain composition and developmental changes were investigated in mice homozygous for the locus "dwarf," and characterized by a reduced level of growth hormone, thyroid stimulating hormone, and prolactin, and by secondary hypothyroidism. The difference in adult brain weight (-32%) between the dwarf and the normal mice was not found to parallel the difference in body weight (-71%), whereas the differences in the weight of the liver (-79%) and that of the kidney (-75%) did. Several biochemical parameters of brain development were assayed in dwarf and normal mice between the ages of 15 and 210 days. Levels of cerebrosides, sulfatides, gangliosides, phospholipids, cholesterol, protein, and RNA (per gram wet weight) were the same for the dwarf and the controls, but the net difference in total brain DNA was less than the net total brain RNA difference (-11% vs. -27%). Total brain lipids (absolute quantities) were the same at 15 days. The difference was -37% by the 50th day, and remained constant thereafter. No change in the specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase or 3'-phosphoadenosine-5'-phosphosulfate: galacto-
This paper is part of the Doctorat d'Etat thesis of L. L. Sarli6ve. Requests for reprints should be addressed to L. L. Sarli6ve, Centre de Neurochimie du CNRS, Facult6 de M6decine, 11 Rue Humann, 67085 Strasbourg Cedex, France. 2 Mr. A. Lees's present address is Harvey Mudd College, Claremont, California 91711. Drs. Sarli~ve and Neskovic are Charg6s de Recherche/~ I'INSERM. 4 Dr. Wintzerith is Charg6e de Recherche au CNRS. Abbreviations used: PAPS: 3'-phosphoadenosine-5'-phosphosulfate; PAPS-CST: 3'phosphoadenosine-5'-phosphosulfate:galactocerebroside suifotransferase; CNP: 2',3'cyclic nucleotide 3'-phosphohydrolase; NeuNAc: N-acetylneuraminic acid.
11
9 1977PlenumPublishingCorporation,227 West 17thStreet, New York, N.Y. 10011. No part of this publicationmay be reproduced, stored in a retrieval system, or transmittedin any form or by any means, electronic, mechanical, photocopying,microfilming,recording,or otherwise,withoutwrittenpermissionof the publisher.
12
LEES ET AL.
cerebroside sulfotransferase was observed. These data suggest that the regulation of the development of brain structures is maintained, but the level of the synthesis of the various brain constituents is reduced in proportion to the brain weight. The development of the dwarf brain seems to proceed harmoniously.
INTRODUCTION The hereditary dwarf mouse of the Black Silver strain was discovered by Snell (1), who concluded that the dwarfism behaves in inheritance as a recessive Mendelian character. Dwarf mature mice are only about one-fourth to one-third the weight of their normal littermates. It is not until the 14th to 16th day, however, that their reduced size becomes obvious. They can usually be identified at this age by their shorter noses and tails, and by their more rounded bodies in comparison with the oblong ones of normal siblings. Their life span under conventional conditions is around 6-8 months, while normal littermates survive for 289 years or more. However, dwarf mice transplanted with lymphocytes from young normal mice survive much longer than untreated ones (2). In addition to juvenile body proportions, dwarf mice show low muscular activity, sterility, and a juvenile type of hair. The developmental peculiarities of the dwarf mouse are due to the reduced pituitary levels of growth hormone (GH) (3-6) and thyroid-stimulating hormone (TSH) (7). A secondary hypothyroidism has also been described (8). Normal growth of dwarf mice can be restored by the administration of either thyroxine or GH or both (2,5,9). In a previous report (10), we studied the DNA, RNA, and protein content of brain, liver, and kidneys in adult dwarf mice and their littermate controls, to evaluate the alteration of growth due to the deficiency in the functioning of the thyroid and the pituitary glands. A striking reduction of DNA and RNA in liver and kidney and a lesser reduction of RNA in brain were observed (10). DNA content was similar in the brains of normal and dwarf mice, while in other organs, the DNA decrease in dwarf mouse was about 70%. Thus, further investigations involving several brain biochemical parameters such as cholesterol, phospholipids, gangliosides, cerebrosides (galactosylceramides), and sulfatides (sulfogalactosylceramides) seemed to be of interest to determine whether the brain development of structures containing these compounds was altered (11). Moreover, it was pertinent to determine whether the alterations found in the brain of the hereditary pituitary dwarf mice were similar to those
B R A I N L I P I D S IN D E V E L O P I N G dw/dw MICE
13
of reported experimentally induced hypothyroidism (12-17) and GH deficiency (18,19). A preliminary report of our studies has been presented (20).
EXPERIMENTAL PROCEDURE
Animals. Mutants, dw/dw (dwarf), or controls with normal phenotype dw/+, + / + , both males and females, and heterozygous breeding pairs of the dwarf Black Silver strain were obtained from the Centre de S~lection et d'Elevage d'Animaux de Laboratoire (Orl6ansla-Source, France). A colony was established in our laboratory and maintained since 1973. The animals were fed a standard diet with water being given ad libitum. Tissue Preparation. Except for nucleic acid determinations, mice were sacrificed by decapitation at 15, 20, 30, 50, and 210 days after birth, and the brains (including the olfactory lobes) were removed immediately, chilled in ice, and weighed. A 10% (wt/vol) homogenate was prepared in 0.05 M Tris-HCl buffer, pH 8.0. An aliquot was reserved for enzyme assays and protein determination, and the remainder was subjected to lipid extraction. Two brains from 15-, 20-, and 30-day-old mice were pooled for each experiment. Each brain from 50-210 day old mice was processed individually. The number of experiments ranged from 3 to 6. All analyses were carried out in duplicate. Protein Determination. Protein was estimated by the method of Lowry et al. (21) using crystalline bovine serum albumin as standard. Nucleic Acid Determination. The R N A and D N A were determined as described previously (10) by the orcinol technique for R N A (22) and the Burton reaction for D N A
(23). Lipid Extraction and Determination. Extraction of the lipids was performed according to the method of Folch et al. (24). Total lipids were measured by weighing the dried lipid extract. Cholesterol (25) and total phospholipids (26) were estimated on the Folch lower phase. The different classes of phospholipids were separated by two-dimensional thinlayer chromatography (TLC) (27), and their phosphorus content was assayed by the method of Dodge and Phillips (28). Cerebrosides and sulfatides were determined by quantitative TLC (29). For some experiments, the technique of Robert and Rebel was used (30). To provide sufficiently large amounts of cerebrosides and sulfatides in the case of 15- and 20-day-old mouse brains, the glycolipid fraction was first purified on a silicic acid column by the method of Vance and Sweeley (31). Estimation of the total gangliosides (NeuNAc) was performed according to the resorcinol procedure of Svennerholm (32), as modified by Miettinen and Takki-Luukkainen (33). T h e individual gangliosides were separated by TLC and quantified by scanning on a Vernon densitometer as previously described (34). Enzyme Assay. The 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.16) was determined by the method of Kurihara and Tsukada (35) after treatment with deoxycholate (36). The reaction product was quantified as previously described (37). The determination of 3'-phosphoadenosine-5'-phosphosulfate : galactocerebroside sulfotransferase (PAPS-CST; EC 2.8.2.11) was carried out according to Sarli6ve et al. (38) with brain homogenate as the source of enzyme.
14
LEES ET AL. RESULTS
Body and Brain Weight During Postnatal Life The body weight of the normal mice increases 4.6-fold between 15 and 210 days of age (Table I). During the same period, there is an increase of only 47% for the dwarf mice. No significant difference was seen at 15 days between the weight of the control and the affected mice. At 20 days, however, the dwarf mice weighed 44% less than the controls, 67% less at 30 days, and 71% less from the 50th to the 210th day. During the period studied, the weight of the normal mouse brain increased, while that of the affected mice remained essentially constant. Beginning at 20 days of age, the dwarf brain always weighs significantly less than the normal. The decrease of the brain weight in dwarf mice compared with controls is 11% at 20 days of age, 20% at 30 days, 27% at 50 days, and 32% at 210 days. Nevertheless, the decrease of the brain weight was always less than that of the body.
Nucleic Acids Table II shows the absolute quantities of RNA and DNA in adult normal and dwarf mice brains. While R N A brain content was 27% lower, the DNA was only 11% lower in the mutant compared with the control. This is also reflected in the significant decrease in the RNA : DNA ratio (-18%), and the significantly increased amount of DNA in the dwarf brain when expressed per unit on a wet weight basis (Table II). As reported previously (10), similar differences were observed for the brain hemispheres and the cerebellum of the dwarf and normal mice.
Protein and Lipids The total protein content of the brain per gram wet weight (relative values) (Table I) increases rapidly until 30 days of age, and then continues to increase more gradually until the protein content of the adult mouse is reached at 210 days. The total lipid content also increases rapidly up to 30 days of age, and then remains constant. The lipid and protein contents are similar in the two types of animals, with only slightly lower values in the mutant brain. However at 50 days of age, the difference is significant for both proteins (-6%) and lipids (-9%) (P
E~ <
CHANGES IN BRAIN COMPONENTS D U R I N G THE D E V E L O P M E N T OF MICE H O M O Z Y G O U S FOR THE L O C U S " D W A R F " (dw) 1'5 A. LEES,2 L. L. SARLII~VE, 3 N. M. NESKOVIC,3 M.
WINTZERITH,
4 AND P.
MANDEL
Centre de Neurochimie du CNRS and Institut de Chimie Biologique FacultO de M~decine I1, Rue Humann, 67085 Strasbourg Cedex, France
Accepted August 13, 1976
Brain composition and developmental changes were investigated in mice homozygous for the locus "dwarf," and characterized by a reduced level of growth hormone, thyroid stimulating hormone, and prolactin, and by secondary hypothyroidism. The difference in adult brain weight (-32%) between the dwarf and the normal mice was not found to parallel the difference in body weight (-71%), whereas the differences in the weight of the liver (-79%) and that of the kidney (-75%) did. Several biochemical parameters of brain development were assayed in dwarf and normal mice between the ages of 15 and 210 days. Levels of cerebrosides, sulfatides, gangliosides, phospholipids, cholesterol, protein, and RNA (per gram wet weight) were the same for the dwarf and the controls, but the net difference in total brain DNA was less than the net total brain RNA difference (-11% vs. -27%). Total brain lipids (absolute quantities) were the same at 15 days. The difference was -37% by the 50th day, and remained constant thereafter. No change in the specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase or 3'-phosphoadenosine-5'-phosphosulfate: galacto-
This paper is part of the Doctorat d'Etat thesis of L. L. Sarli6ve. Requests for reprints should be addressed to L. L. Sarli6ve, Centre de Neurochimie du CNRS, Facult6 de M6decine, 11 Rue Humann, 67085 Strasbourg Cedex, France. 2 Mr. A. Lees's present address is Harvey Mudd College, Claremont, California 91711. Drs. Sarli~ve and Neskovic are Charg6s de Recherche/~ I'INSERM. 4 Dr. Wintzerith is Charg6e de Recherche au CNRS. Abbreviations used: PAPS: 3'-phosphoadenosine-5'-phosphosulfate; PAPS-CST: 3'phosphoadenosine-5'-phosphosulfate:galactocerebroside suifotransferase; CNP: 2',3'cyclic nucleotide 3'-phosphohydrolase; NeuNAc: N-acetylneuraminic acid.
11
9 1977PlenumPublishingCorporation,227 West 17thStreet, New York, N.Y. 10011. No part of this publicationmay be reproduced, stored in a retrieval system, or transmittedin any form or by any means, electronic, mechanical, photocopying,microfilming,recording,or otherwise,withoutwrittenpermissionof the publisher.
12
LEES ET AL.
cerebroside sulfotransferase was observed. These data suggest that the regulation of the development of brain structures is maintained, but the level of the synthesis of the various brain constituents is reduced in proportion to the brain weight. The development of the dwarf brain seems to proceed harmoniously.
INTRODUCTION The hereditary dwarf mouse of the Black Silver strain was discovered by Snell (1), who concluded that the dwarfism behaves in inheritance as a recessive Mendelian character. Dwarf mature mice are only about one-fourth to one-third the weight of their normal littermates. It is not until the 14th to 16th day, however, that their reduced size becomes obvious. They can usually be identified at this age by their shorter noses and tails, and by their more rounded bodies in comparison with the oblong ones of normal siblings. Their life span under conventional conditions is around 6-8 months, while normal littermates survive for 289 years or more. However, dwarf mice transplanted with lymphocytes from young normal mice survive much longer than untreated ones (2). In addition to juvenile body proportions, dwarf mice show low muscular activity, sterility, and a juvenile type of hair. The developmental peculiarities of the dwarf mouse are due to the reduced pituitary levels of growth hormone (GH) (3-6) and thyroid-stimulating hormone (TSH) (7). A secondary hypothyroidism has also been described (8). Normal growth of dwarf mice can be restored by the administration of either thyroxine or GH or both (2,5,9). In a previous report (10), we studied the DNA, RNA, and protein content of brain, liver, and kidneys in adult dwarf mice and their littermate controls, to evaluate the alteration of growth due to the deficiency in the functioning of the thyroid and the pituitary glands. A striking reduction of DNA and RNA in liver and kidney and a lesser reduction of RNA in brain were observed (10). DNA content was similar in the brains of normal and dwarf mice, while in other organs, the DNA decrease in dwarf mouse was about 70%. Thus, further investigations involving several brain biochemical parameters such as cholesterol, phospholipids, gangliosides, cerebrosides (galactosylceramides), and sulfatides (sulfogalactosylceramides) seemed to be of interest to determine whether the brain development of structures containing these compounds was altered (11). Moreover, it was pertinent to determine whether the alterations found in the brain of the hereditary pituitary dwarf mice were similar to those
B R A I N L I P I D S IN D E V E L O P I N G dw/dw MICE
13
of reported experimentally induced hypothyroidism (12-17) and GH deficiency (18,19). A preliminary report of our studies has been presented (20).
EXPERIMENTAL PROCEDURE
Animals. Mutants, dw/dw (dwarf), or controls with normal phenotype dw/+, + / + , both males and females, and heterozygous breeding pairs of the dwarf Black Silver strain were obtained from the Centre de S~lection et d'Elevage d'Animaux de Laboratoire (Orl6ansla-Source, France). A colony was established in our laboratory and maintained since 1973. The animals were fed a standard diet with water being given ad libitum. Tissue Preparation. Except for nucleic acid determinations, mice were sacrificed by decapitation at 15, 20, 30, 50, and 210 days after birth, and the brains (including the olfactory lobes) were removed immediately, chilled in ice, and weighed. A 10% (wt/vol) homogenate was prepared in 0.05 M Tris-HCl buffer, pH 8.0. An aliquot was reserved for enzyme assays and protein determination, and the remainder was subjected to lipid extraction. Two brains from 15-, 20-, and 30-day-old mice were pooled for each experiment. Each brain from 50-210 day old mice was processed individually. The number of experiments ranged from 3 to 6. All analyses were carried out in duplicate. Protein Determination. Protein was estimated by the method of Lowry et al. (21) using crystalline bovine serum albumin as standard. Nucleic Acid Determination. The R N A and D N A were determined as described previously (10) by the orcinol technique for R N A (22) and the Burton reaction for D N A
(23). Lipid Extraction and Determination. Extraction of the lipids was performed according to the method of Folch et al. (24). Total lipids were measured by weighing the dried lipid extract. Cholesterol (25) and total phospholipids (26) were estimated on the Folch lower phase. The different classes of phospholipids were separated by two-dimensional thinlayer chromatography (TLC) (27), and their phosphorus content was assayed by the method of Dodge and Phillips (28). Cerebrosides and sulfatides were determined by quantitative TLC (29). For some experiments, the technique of Robert and Rebel was used (30). To provide sufficiently large amounts of cerebrosides and sulfatides in the case of 15- and 20-day-old mouse brains, the glycolipid fraction was first purified on a silicic acid column by the method of Vance and Sweeley (31). Estimation of the total gangliosides (NeuNAc) was performed according to the resorcinol procedure of Svennerholm (32), as modified by Miettinen and Takki-Luukkainen (33). T h e individual gangliosides were separated by TLC and quantified by scanning on a Vernon densitometer as previously described (34). Enzyme Assay. The 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.16) was determined by the method of Kurihara and Tsukada (35) after treatment with deoxycholate (36). The reaction product was quantified as previously described (37). The determination of 3'-phosphoadenosine-5'-phosphosulfate : galactocerebroside sulfotransferase (PAPS-CST; EC 2.8.2.11) was carried out according to Sarli6ve et al. (38) with brain homogenate as the source of enzyme.
14
LEES ET AL. RESULTS
Body and Brain Weight During Postnatal Life The body weight of the normal mice increases 4.6-fold between 15 and 210 days of age (Table I). During the same period, there is an increase of only 47% for the dwarf mice. No significant difference was seen at 15 days between the weight of the control and the affected mice. At 20 days, however, the dwarf mice weighed 44% less than the controls, 67% less at 30 days, and 71% less from the 50th to the 210th day. During the period studied, the weight of the normal mouse brain increased, while that of the affected mice remained essentially constant. Beginning at 20 days of age, the dwarf brain always weighs significantly less than the normal. The decrease of the brain weight in dwarf mice compared with controls is 11% at 20 days of age, 20% at 30 days, 27% at 50 days, and 32% at 210 days. Nevertheless, the decrease of the brain weight was always less than that of the body.
Nucleic Acids Table II shows the absolute quantities of RNA and DNA in adult normal and dwarf mice brains. While R N A brain content was 27% lower, the DNA was only 11% lower in the mutant compared with the control. This is also reflected in the significant decrease in the RNA : DNA ratio (-18%), and the significantly increased amount of DNA in the dwarf brain when expressed per unit on a wet weight basis (Table II). As reported previously (10), similar differences were observed for the brain hemispheres and the cerebellum of the dwarf and normal mice.
Protein and Lipids The total protein content of the brain per gram wet weight (relative values) (Table I) increases rapidly until 30 days of age, and then continues to increase more gradually until the protein content of the adult mouse is reached at 210 days. The total lipid content also increases rapidly up to 30 days of age, and then remains constant. The lipid and protein contents are similar in the two types of animals, with only slightly lower values in the mutant brain. However at 50 days of age, the difference is significant for both proteins (-6%) and lipids (-9%) (P
E~ <
