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Whole brain DNA in normal and abnormal embryos of the loop-tail (Lp) mutant mouse
354
Brain Research, 140 (1978) 354 359 ;(? Elsevier/North-Holland Biomedical Press
Whole brain DNA in normal and abnormal embryos of the loop-tail mutant mouse
(Lp)
DORIS B. WILSON and LINDA W. GONZALES Division of Anatomy, M-O04, Department of Surgery, University of California, San Diego, School of Medicine, La Jolla, Calif. 92093 and Department of Neurology, School of Medicine, University of California, Davis, Calif. 95616 (U.S.A.) (Accepted August 24th, 1977)
The loop-tail (Lp) mutant mouse has been of interest to various investigators because of the consistent and severe neural tube defects exhibited by homozygous (Lp/Lp) embryos 22,26,27,a°. These defects include rachischisis which is characterized by an open neural tube from the caudal midbrain area to the tail. Axial skeletal abnormalities are also associated with the neural defect 25. Although the homozygous loop-tail embryos die at birth, heterozygous (Lp/+) individuals are fully viable and show only mild morphological effects of the Lp gene, i.e., varying degrees of loops or kinks in the tail. Early studies on this mutant described an 'overgrowth' characterized by increased numbers of mitotic figures in the neural tissue of midbrain and hindbrain of the homozygotes (Lp/Lp) 24-26. However, quantitative autoradiographic studies on the cell cycle during the 10th and 1 lth days of gestation have shown that there is actually a lengthening in the total generation time of the cycle, especially in the duration of the mitotic (M) phase, suggesting that this increase in mitotic figures is not due to increased proliferation but rather to the prolongation of the M phase zg. Disturbances in cellular proliferation during development have been shown to have an effect on cell number 1,6,9,12,14-16,1a-z°,2a,81'32. In many of these studies measurements of DNA have been used as an index of cell number, since the amount of DNA/nucleus remains relatively constant for each species 3,7,1°,z8. The current study was thus undertaken in order to determine whether the early proliferative defects in the abnormal loop-tail (Lp/Lp) mice produce any detectable differences in DNA content as compared with that in their normal ( ÷ / + ; L p / + ) littermates. Measurements were made of wet weight, total #g DNA,/~g DNA/mg tissue,/~g protein/rag tissue, and Fg protein/#g DNA in whole brains of loop-tail embryos at 15 days and at 18 days of gestation. The loop-tail mice used in this study have been derived from an inbred line obtained from Dr. Kathryn F. Stein at Mount Holyoke College. The animals were maintained on a light-dark cycle (14 h light, 10 h dark) with a diet of Purina Mouse Chow. Timed embryos were obtained from Lp/ q- × L p / + matings, with day 0 being
355 the day on which the vaginal plug was observed. On either day 15 or day 18, embryos were removed and identified as normals ( + / + ; L p / + ) or abnormals (Lp/Lp). The brains were dissected free of surrounding tissues, blotted on filter paper, weighed and immediately frozen. A total of 17 normal and 4 abnormal brains was used for the 15day group, and 17 normal and 3 abnormals for the 18-day group. Each brain was extracted with 20 vol of cold 0.6 N perchloric acid by a 5-6 sec sonication burst at setting 1 (Branson Sonifier), left on ice for 1 h, and centrifuged at 12,000 × g for 10 min at 4 °C. The precipitate was washed with an equal volume of 0.2 N perchloric acid and centrifuged as above. The acid-insoluble precipitate was extracted with 5 ~ trichloroacetic acid (TCA) at 90 °C for 15 min, cooled in ice, and centrifuged as above. The residue was washed twice by resuspension in cold 5 ~ TCA and centrifuged. The last three supernatants were combined and retained for D N A analysis. D N A was determined by the diphenylamine method of Burton 4 as modified by Geel and Timiras 11 using calf thymus D N A (Sigma Chemical, St. Louis, Mo.) as the standard. The precipitates remaining after TCA-extraction were washed twice with cold ether, centrifuged, and residual ether removed under N2. The pellets were solubilized in 0.5 N N a O H at 37 °C for 18 h and protein determined by the method of Lowry et al. 17 using bovine serum albumin (Sigma) as the standard. Values for the normal and abnormal brains were expressed as means 4- S.E.M. Significance of differences between the values was determined by the Student t-test, with P < 0.05 being significant. In this study the term normal is applied to homozygous normal ( + / + ) and heterozygous loop-tail ( L p / + ) embryos. Abnormal refers to homozygous loop-tail (Lp/Lp). A summary of the results is presented in Table I. Wet weight. The mean wet weight of whole brains from the normal 15-day embryos was 45.2 4- 4.1 mg; in the abnormals it was 34.9 ± 2.1 mg. This difference was significant (P < 0.01), and the abnormal weight was approximately 77 ~ of the normals. TABLE I
Comparison of normal (q-/+ ; Lp/+) and abnormal (Lp/Lp) brains at 15- and 18-day gestation Values for brain weight (mg), total DNA ~g), ~g DNA/mg tissue,/~g protein/mgtissue, and pg protein/ pg DNA are expressed as means + S.E.M., and abnormal brain values which are significantlydifferent from normal brain values at the same age are indicated by *P < 0.01, **P < 0.05.
15-day Normal (+/+;Lp/+) Brain weight (mg) Total DNA Q~g) /~g DNA/mg tissue /tg protein/rag tissue k,g protein/#g DNA
18-day Abnormal (Lp/Lp)
Normal (+/+;Lp/+)
Abnormal (Lp/Lp)
45.2 ± 4.1 34.9 5- 2.1" 83.5 ± 1.5 78.8 ± 5.1 265.1 4- 6.8 262.2 -q- 11.9 351.8 ± 7.9 352.9 -4- 43.7 5.860 i 0.147 7.5504- 0.311" 4.236 i 0.121 4.4744- 0.338 69.85 4- 0.84 79.03 ± 1.82" 85.954- 2.72 89.40 -I- 7.54 12.018 4- 0.244 10.486 4- 0.346** 20.392 4- 0.147 19.956 4- 0.290
356 By 18 days the mean weight of normal whole brain was 83.5 4- 1.5 mg. In the abnormals it was 78.8 4- 5.1 mg, or 9 4 ~ of the normals. This difference was not significant (P > 0.20). Total DNA. At 15 days the mean total DNA in the normal brains was 265.1 46.8/~g, while that in the abnormal brains was 262.2 ± 11.9/~g, a difference which was not significant (P > 0.50). At 18 days the difference was also not significant ( P ) - 0.50; normal = 351.8 ± 7.9 pg; abnormal = 352.9 ± 43.7 #g). DNA/tissue. A significant difference (P < 0.01) occurred in the tissue concentration of DNA between normal and abnormal brains at 15 days. The normal brains showed 5.860 4- 0.147 #g/mg tissue, as compared with 7.550 4- 0.311 #g/rag tissue in the abnormals. However, by 18 days the difference was not significant (P > 0.20), with a normal mean value of 4.236 4- 0.121 #g/mg tissue and an abnormal mean value of 4.474 4- 0.338 #g/mg tissue. Protein~tissue. At 15 days of gestation the abnormal brains showed a higher protein/tissue ratio (79.03 4- 1.82/~g/mg tissue) than did the normal brains (69.85 4- 0.84 #g/mg tissue). This difference was significant (P < 0.01). Although the 18-day abnormal embryos likewise showed a higher ratio (89.40 4- 7.54 pg/mg tissue) than did their normal littermates (85.95 4- 2.72 #g/mg tissue), this difference was not significant (P > 0.50). Protein/DNA. The ratio of protein/DNA was much lower in the 15-day abnormal brains (10.486 4- 0.346 #g protein//~g DNA) than in the normal brains (12.018 4- 0.244 pg protein/#g DNA), the difference being significant (P < 0.05). In contrast, the ratios in the 18-day embryos were not significant (P > 0.20) (normal = 20.392 4- 0.147/~g protein/#g DNA; abnormal = 19.956 4- 0.290 ~g protein/#g DNA). Since determinations of absolute values of DNA in brain tissue may differ depending on variations in the assay method used 8a, the results of the present study are of use mainly in analyzing relative amounts of DNA content in normal and abnormal brains. Moreover no attempt was made to calculate actual cell numbers for these brains, but rather to use comparisons of total DNA as indices of relative cell number in the normals and abnormals. It is of interest that all of the parameters studied at 15 days' gestation showed highly significant differences between the normal and abnormal brains, except for mean total DNA content (normal = 265.1 /~g: abnormal -- 262.2 #g). This similarity in total DNA suggests that the abnormal brains contained approximately the same number of cells as did the normal brains. Since the wet weight of the abnormal brains was significantly less than that of the normal brains, the mean concentration of DNA/mg tissue was higher in the abnormal brains (7.550/~g/mg as opposed to 5.860 #g/mg), suggesting that the abnormal brain cells were smaller. The fact that the protein/DNA ratio was lower in the abnormals (abnormal ~ 10.486; normal = 12.018) likewise implies a smaller cell size in the abnormal brains. A similar reduction in brain weight due to a decrease in cell size rather than to a decrease in cell number has been observed in neonatally thyroidectomized rats 2,s,11. Also, in dwarf
357 mice the amount of DNA/spinal cord was found to be the same as in their normal littermates, while lower protein/DNA indicated a reduction in cell size21. It is difficult to interpret the significance of the higher amount of protein/mg tissue in the abnormal brains than in the normal ones, since determinations were not made of water content or of the relative amounts of other cellular and extracellular constituents such as lipids. However, the lack of any increase in total amount of DNA (and thus in cell number) is important, since this corroborates earlier autoradiographic data that the increased number of mitotic figures characteristically seen in the 10- and I 1-day abnormal brain does not represent an increase in mitotic activity2L Also, attempts to count and tabulate numbers of cells in the hindbrain of abnormal looptails have failed to show consistent increases in total cell number 24. Consequently there is no true 'overgrowth' or increased proliferation of the neural tissue, as has been previously described in these abnormal embryos13,~5,z6. Since cellular kinetics studies have shown that the increased mitotic figures are actually due to a prolongation in the duration of the mitotic phase of the cell cycle, as well as in G1, and in the total generation time 29, one would have expected to find a decrease in total DNA in the abnormal brains, rather that the same amount of DNA as in the normal controls. However, the prolongation of the generation time was found to be only 4-5 h in the abnormal brains 29, and it is possible that this delay might not be sufficient to exert such a dramatic effect on cell number as to be detected in later stages by biochemical assay. It is also possible that, between the 12th and 15th day of gestation, the neural cells in abnormal brains retain their proliferative capacities longer than do those in normal brains, thus enabling them to restore any deficits in cell number by the 15th day. This possibility is currently being investigated autoradiographically. The effects of the Lp gene in the homozygous condition are somewhat different from those induced experimentally by various chemicals or by nutritional intervention. For example some agents retard growth in rat fetuses resulting in a decrease in total fetal DNA and cell number 6. Similar reductions have been observed subsequent to prenatal treatment with methylazoxymethanoPs, and in response to prenatal hyperthermia9 and to postnatal hyperoxia12. Reductions in cell number have also been achieved in mice following corticosterone treatment14,15, and in rats subsequent to maternal dietary protein restrictiona~, 82. Moreover, growth retardation in human embryos has been shown to result in a decrease in brain weight and in total DNA~. The reduced brain weight and protein/DNA observed in the 15-day loop-tail homozygote are of interest because they are eventually restored to normal values by 18 days' gestation. The manner in which these cells are able to catch up with their normal counterparts deserves further attention, particularly in terms of changes in their fine structure and biochemical composition between the 15th and 18th day of gestation. This research was supported by National Institutes of Health Research Grant HD 09562 from the National Institute of Child Health and Human Development. The authors wish to thank Dr. Stanley E. Geel for his advice and suggestions concerning the biochemical techniques.
358 1 Balazs, R. and Cotterrell, M., Effect of hormonal state on cell number and functional maturation of the brain, Nature (Lond.), 236 (1972) 348-350. 2 Balazs, R., Kovacs, S., Teichgraber, P., Cocks, W. A., and Eayrs, J. T., Biochemical effects of thyroid deficiency on the developing brain, J. Neurochem., 15 (1968) 1335-1349. 3 Boivin, A., Vendrely, R. and Vendrely, C., L'acide d6oxyribonucl6ique du noyau cellulaire d6positaire des caract+res h6r6ditaires; arguments d'ordre analytique, C.R. Acad. Sci., 226 (1948) 1061-1063. 4 Burton, K., A study of the conditions and mechanism of the diphenylamine reaction for tl-,e colorimetric estimation of DNA, Biochem. J., 62 (1956) 315-323. 5 Chase, H. P., Welch, N. N., Dabiere, C. S., Vasan, N. S. and Butterfield, L. J., Alterations in human brain biochemistry following intrauterine growth retardation, Pediatrics, 50 (1972) 403-41 I. 6 Chaube, S. and Swinyard, C. A., Cellular and biochemical aspects of growth retardation in rat fetuses induced by maternal administration of selected anticancer agents, Teratology, 12 (1975) 259-270. 7 Davidson, J. N. and Leslie, I., Nucleic acid in relation to tissue growth, Cancer Res., 10 (1950) 587-594. 8 Eayrs, J. T. and Taylor, S. H., The effects of thyroid deficiency induced by methyl thiouracil on the maturation of the central nervous system, J. Anat. (Lond.), 85 (1951) 356-358. 9 Edwards, M. J., Penny, R. H. and Zevnik, J., A brain cell deficit in newborn guinea-pigs following prenatal hyperthermia, Brain Research, 28 (1971) 341-345. 10 Enesco, M. and Leblond, C. P., Increase in cell number as a factor in the growth of the organs of the young male rat, J. Embryol. exp. Morphol., 10 (1962) 530-562. 11 Geel, S. E. and Timiras, P. S., The influence of neonatal hypothyroidism and of thyroxine on the ribonucleic acid and deoxyribonucleic acid concentrations of rat cerebral cortex, Brain Research, 4 (1967) 135-142. 12 Grave, G. D., Kennedy, C. and Sokoloff, L., Impairment of growth and development of the rat brain by hyperoxia at atmospheric pressure, J. Neurochem., 19 (1972) 187-194. 13 Grfineberg, H., The Pathology of Development, John Wiley, New York, 1963, p. 154. 14 Howard, E., Effects of corticosterone and food restriction on growth and on DNA, RNA and cholesterol contents of the brain and liver in infant mice, J. Neurochem., 12 (1965) 181-191. 15 Howard, E., Reductions in size and total D N A of cerebrum and cerebellum in adult mice after corticosterone treatment in infancy, Exp. Neurol., 22 (1968) 191-208. 16 Lewis, P. D., Balazs, R., Patel, A. J. and Johnson, A. L., The effect of undernutrition in early life on cell generation in the rat brain, Brain Research, 83 (1975) 235-247. 17 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, J. R., Protein measurement with tt',e Folin-phenol reagent, J. bioL Chem., 193 (1951) 265-275. 18 Matsumoto, H., Spatz, M. and Laqueur, G. L., Quantitative changes with age in the D N A content of methylazoxymethanol-induced microencephalic rat brain, J. Neurochem., 19 (1972) 297-306 19 Patel, A. J., Balazs, R., and Johnson, A. L., Effect of undernutrition on cell formation in the rat brain, J. Neurochem., 20 (1973) 1151-1165. 20 Petropoulos, E. A., Vernadakis, A. and Timiras, P. S., Nucleic acid content in developing rat brain after prenatal and/or neonatal exposure to high altitude, Fed. Proc., 28 (1969) lC01-1005. 21 Sawchak, J. A., Postnatal spinal cord development in dwarf (Snell's) mice, Anat. Rec., 178 (1974) 456. 22 Smith, L. J. and Stein, K. F., Axial elongation in the mouse and its retardation in homozygous looptail mice, J. Embryol. exp. MorphoL, 10 (1962) 73-87. 23 Stastny, F. and Svoboda, J., Influence of hydrocortisor, e on DNA, R N A and protein in the chick embryo cerebral hemispheres, Acta Univ. Carol. Med. (Praha), 18 (1972) 229-242. 24 Stein, K. F., Lievre, F. and Smoller, C. G., Abnormal brain differentiation in the homozygcus loop-tail embryo, Anat. Rec., 136 (1960)324-325. 25 Stein, K. F. and Mackensen, J. A., Abnormal development of the thoracic skeleton in mice homczygous for the gene for looped-tail, Amer. J. Anat., 100 (1957) 205-241. 26 Stein, K. F. and Rudin, I. A., Development of mice homozygous for the gene for looptail, J. Hered., 44 (1953) 59-69. 27 Strong, L. C. and Hollander, W. F., Hereditary loop-tail in the house mouse, J. Hered., 40 (1949) 329-334. 28 Vendrely, R. and Vendrely, C., La teneur du noyau cellulaire en acide d6soxyribonucl6ique a travers des organs des individus et les esp~ces animals, Experientia (Basel), 4 (1948) 4~4-436.
359 29 Wilson, D. B. and Center, E. M., The neural cell cycle in the looptail (Lp) mutant mouse, J. EmbryoL exp. Morphol., 32 (1974) 697-705. 30 Wilson, D. B. and Michael, S. D., Surface defects in ventricular cells of brains of mouse embryos homozygous for the loop-tail gene: scanning electron microscopic study, Teratology, 11 (1975) 87-98. 31 Zamenhof, S., Van Marthens, E. and Grauel, L., DNA (cell number) and protein in neonatal rat brain: alteration by timing of maternal dietary protein restriction, J. Nutr., 101 (1971) 1265-1269. 32 Zamenhof, S., Van Marthens, E. and Grauel, L., DNA (cell number) and protein in rat brain. Second generation (F2) alteration by maternal (F0) dietary protein restriction, Nutr. Metab., 14 (1972) 262-270. 33 Zamenhof, S., Bursztyn, H., Rich, K. and Zamenhof, P., The determination of deoxyribonucleic acid and of cell number in brain, J. Neurochem., 11 (1964) 505-509.
Brain Research, 140 (1978) 354 359 ;(? Elsevier/North-Holland Biomedical Press
Whole brain DNA in normal and abnormal embryos of the loop-tail mutant mouse
(Lp)
DORIS B. WILSON and LINDA W. GONZALES Division of Anatomy, M-O04, Department of Surgery, University of California, San Diego, School of Medicine, La Jolla, Calif. 92093 and Department of Neurology, School of Medicine, University of California, Davis, Calif. 95616 (U.S.A.) (Accepted August 24th, 1977)
The loop-tail (Lp) mutant mouse has been of interest to various investigators because of the consistent and severe neural tube defects exhibited by homozygous (Lp/Lp) embryos 22,26,27,a°. These defects include rachischisis which is characterized by an open neural tube from the caudal midbrain area to the tail. Axial skeletal abnormalities are also associated with the neural defect 25. Although the homozygous loop-tail embryos die at birth, heterozygous (Lp/+) individuals are fully viable and show only mild morphological effects of the Lp gene, i.e., varying degrees of loops or kinks in the tail. Early studies on this mutant described an 'overgrowth' characterized by increased numbers of mitotic figures in the neural tissue of midbrain and hindbrain of the homozygotes (Lp/Lp) 24-26. However, quantitative autoradiographic studies on the cell cycle during the 10th and 1 lth days of gestation have shown that there is actually a lengthening in the total generation time of the cycle, especially in the duration of the mitotic (M) phase, suggesting that this increase in mitotic figures is not due to increased proliferation but rather to the prolongation of the M phase zg. Disturbances in cellular proliferation during development have been shown to have an effect on cell number 1,6,9,12,14-16,1a-z°,2a,81'32. In many of these studies measurements of DNA have been used as an index of cell number, since the amount of DNA/nucleus remains relatively constant for each species 3,7,1°,z8. The current study was thus undertaken in order to determine whether the early proliferative defects in the abnormal loop-tail (Lp/Lp) mice produce any detectable differences in DNA content as compared with that in their normal ( ÷ / + ; L p / + ) littermates. Measurements were made of wet weight, total #g DNA,/~g DNA/mg tissue,/~g protein/rag tissue, and Fg protein/#g DNA in whole brains of loop-tail embryos at 15 days and at 18 days of gestation. The loop-tail mice used in this study have been derived from an inbred line obtained from Dr. Kathryn F. Stein at Mount Holyoke College. The animals were maintained on a light-dark cycle (14 h light, 10 h dark) with a diet of Purina Mouse Chow. Timed embryos were obtained from Lp/ q- × L p / + matings, with day 0 being
355 the day on which the vaginal plug was observed. On either day 15 or day 18, embryos were removed and identified as normals ( + / + ; L p / + ) or abnormals (Lp/Lp). The brains were dissected free of surrounding tissues, blotted on filter paper, weighed and immediately frozen. A total of 17 normal and 4 abnormal brains was used for the 15day group, and 17 normal and 3 abnormals for the 18-day group. Each brain was extracted with 20 vol of cold 0.6 N perchloric acid by a 5-6 sec sonication burst at setting 1 (Branson Sonifier), left on ice for 1 h, and centrifuged at 12,000 × g for 10 min at 4 °C. The precipitate was washed with an equal volume of 0.2 N perchloric acid and centrifuged as above. The acid-insoluble precipitate was extracted with 5 ~ trichloroacetic acid (TCA) at 90 °C for 15 min, cooled in ice, and centrifuged as above. The residue was washed twice by resuspension in cold 5 ~ TCA and centrifuged. The last three supernatants were combined and retained for D N A analysis. D N A was determined by the diphenylamine method of Burton 4 as modified by Geel and Timiras 11 using calf thymus D N A (Sigma Chemical, St. Louis, Mo.) as the standard. The precipitates remaining after TCA-extraction were washed twice with cold ether, centrifuged, and residual ether removed under N2. The pellets were solubilized in 0.5 N N a O H at 37 °C for 18 h and protein determined by the method of Lowry et al. 17 using bovine serum albumin (Sigma) as the standard. Values for the normal and abnormal brains were expressed as means 4- S.E.M. Significance of differences between the values was determined by the Student t-test, with P < 0.05 being significant. In this study the term normal is applied to homozygous normal ( + / + ) and heterozygous loop-tail ( L p / + ) embryos. Abnormal refers to homozygous loop-tail (Lp/Lp). A summary of the results is presented in Table I. Wet weight. The mean wet weight of whole brains from the normal 15-day embryos was 45.2 4- 4.1 mg; in the abnormals it was 34.9 ± 2.1 mg. This difference was significant (P < 0.01), and the abnormal weight was approximately 77 ~ of the normals. TABLE I
Comparison of normal (q-/+ ; Lp/+) and abnormal (Lp/Lp) brains at 15- and 18-day gestation Values for brain weight (mg), total DNA ~g), ~g DNA/mg tissue,/~g protein/mgtissue, and pg protein/ pg DNA are expressed as means + S.E.M., and abnormal brain values which are significantlydifferent from normal brain values at the same age are indicated by *P < 0.01, **P < 0.05.
15-day Normal (+/+;Lp/+) Brain weight (mg) Total DNA Q~g) /~g DNA/mg tissue /tg protein/rag tissue k,g protein/#g DNA
18-day Abnormal (Lp/Lp)
Normal (+/+;Lp/+)
Abnormal (Lp/Lp)
45.2 ± 4.1 34.9 5- 2.1" 83.5 ± 1.5 78.8 ± 5.1 265.1 4- 6.8 262.2 -q- 11.9 351.8 ± 7.9 352.9 -4- 43.7 5.860 i 0.147 7.5504- 0.311" 4.236 i 0.121 4.4744- 0.338 69.85 4- 0.84 79.03 ± 1.82" 85.954- 2.72 89.40 -I- 7.54 12.018 4- 0.244 10.486 4- 0.346** 20.392 4- 0.147 19.956 4- 0.290
356 By 18 days the mean weight of normal whole brain was 83.5 4- 1.5 mg. In the abnormals it was 78.8 4- 5.1 mg, or 9 4 ~ of the normals. This difference was not significant (P > 0.20). Total DNA. At 15 days the mean total DNA in the normal brains was 265.1 46.8/~g, while that in the abnormal brains was 262.2 ± 11.9/~g, a difference which was not significant (P > 0.50). At 18 days the difference was also not significant ( P ) - 0.50; normal = 351.8 ± 7.9 pg; abnormal = 352.9 ± 43.7 #g). DNA/tissue. A significant difference (P < 0.01) occurred in the tissue concentration of DNA between normal and abnormal brains at 15 days. The normal brains showed 5.860 4- 0.147 #g/mg tissue, as compared with 7.550 4- 0.311 #g/rag tissue in the abnormals. However, by 18 days the difference was not significant (P > 0.20), with a normal mean value of 4.236 4- 0.121 #g/mg tissue and an abnormal mean value of 4.474 4- 0.338 #g/mg tissue. Protein~tissue. At 15 days of gestation the abnormal brains showed a higher protein/tissue ratio (79.03 4- 1.82/~g/mg tissue) than did the normal brains (69.85 4- 0.84 #g/mg tissue). This difference was significant (P < 0.01). Although the 18-day abnormal embryos likewise showed a higher ratio (89.40 4- 7.54 pg/mg tissue) than did their normal littermates (85.95 4- 2.72 #g/mg tissue), this difference was not significant (P > 0.50). Protein/DNA. The ratio of protein/DNA was much lower in the 15-day abnormal brains (10.486 4- 0.346 #g protein//~g DNA) than in the normal brains (12.018 4- 0.244 pg protein/#g DNA), the difference being significant (P < 0.05). In contrast, the ratios in the 18-day embryos were not significant (P > 0.20) (normal = 20.392 4- 0.147/~g protein/#g DNA; abnormal = 19.956 4- 0.290 ~g protein/#g DNA). Since determinations of absolute values of DNA in brain tissue may differ depending on variations in the assay method used 8a, the results of the present study are of use mainly in analyzing relative amounts of DNA content in normal and abnormal brains. Moreover no attempt was made to calculate actual cell numbers for these brains, but rather to use comparisons of total DNA as indices of relative cell number in the normals and abnormals. It is of interest that all of the parameters studied at 15 days' gestation showed highly significant differences between the normal and abnormal brains, except for mean total DNA content (normal = 265.1 /~g: abnormal -- 262.2 #g). This similarity in total DNA suggests that the abnormal brains contained approximately the same number of cells as did the normal brains. Since the wet weight of the abnormal brains was significantly less than that of the normal brains, the mean concentration of DNA/mg tissue was higher in the abnormal brains (7.550/~g/mg as opposed to 5.860 #g/mg), suggesting that the abnormal brain cells were smaller. The fact that the protein/DNA ratio was lower in the abnormals (abnormal ~ 10.486; normal = 12.018) likewise implies a smaller cell size in the abnormal brains. A similar reduction in brain weight due to a decrease in cell size rather than to a decrease in cell number has been observed in neonatally thyroidectomized rats 2,s,11. Also, in dwarf
357 mice the amount of DNA/spinal cord was found to be the same as in their normal littermates, while lower protein/DNA indicated a reduction in cell size21. It is difficult to interpret the significance of the higher amount of protein/mg tissue in the abnormal brains than in the normal ones, since determinations were not made of water content or of the relative amounts of other cellular and extracellular constituents such as lipids. However, the lack of any increase in total amount of DNA (and thus in cell number) is important, since this corroborates earlier autoradiographic data that the increased number of mitotic figures characteristically seen in the 10- and I 1-day abnormal brain does not represent an increase in mitotic activity2L Also, attempts to count and tabulate numbers of cells in the hindbrain of abnormal looptails have failed to show consistent increases in total cell number 24. Consequently there is no true 'overgrowth' or increased proliferation of the neural tissue, as has been previously described in these abnormal embryos13,~5,z6. Since cellular kinetics studies have shown that the increased mitotic figures are actually due to a prolongation in the duration of the mitotic phase of the cell cycle, as well as in G1, and in the total generation time 29, one would have expected to find a decrease in total DNA in the abnormal brains, rather that the same amount of DNA as in the normal controls. However, the prolongation of the generation time was found to be only 4-5 h in the abnormal brains 29, and it is possible that this delay might not be sufficient to exert such a dramatic effect on cell number as to be detected in later stages by biochemical assay. It is also possible that, between the 12th and 15th day of gestation, the neural cells in abnormal brains retain their proliferative capacities longer than do those in normal brains, thus enabling them to restore any deficits in cell number by the 15th day. This possibility is currently being investigated autoradiographically. The effects of the Lp gene in the homozygous condition are somewhat different from those induced experimentally by various chemicals or by nutritional intervention. For example some agents retard growth in rat fetuses resulting in a decrease in total fetal DNA and cell number 6. Similar reductions have been observed subsequent to prenatal treatment with methylazoxymethanoPs, and in response to prenatal hyperthermia9 and to postnatal hyperoxia12. Reductions in cell number have also been achieved in mice following corticosterone treatment14,15, and in rats subsequent to maternal dietary protein restrictiona~, 82. Moreover, growth retardation in human embryos has been shown to result in a decrease in brain weight and in total DNA~. The reduced brain weight and protein/DNA observed in the 15-day loop-tail homozygote are of interest because they are eventually restored to normal values by 18 days' gestation. The manner in which these cells are able to catch up with their normal counterparts deserves further attention, particularly in terms of changes in their fine structure and biochemical composition between the 15th and 18th day of gestation. This research was supported by National Institutes of Health Research Grant HD 09562 from the National Institute of Child Health and Human Development. The authors wish to thank Dr. Stanley E. Geel for his advice and suggestions concerning the biochemical techniques.
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