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Zinc deficiency in the 11 day rat embryo: A scanning and transmission electron microscope study
Life Sciences, Vol. 42, pp. 889-896 Printed in the U.S.A.
Pergamon Journals
ZINC DEFICIENCY IN THE 11 DAY RAT EMBRYO: A SCANNING AND TRANSMISSION ELECTRON MICROSCOPE STUDY 2 A.J. Harding 192 , I.E. Dreosti 193 , and R.S. Tulsi .
1 2 3
CSIRO Division of Human Nutrition, Adelaide, Australia. Department of Anatomy and Histology, Adelaide Univeristy, Adelaide, Australia. To whom correspondence should be addressed. (Received in final form December 18, 1987)
Summary Zinc deficient rat embryos were obtained on the 11th day of pregnancy and examined by scanning and transmission electron microscopy. Scanning electron microscopy revealed an increase in the number of deformed embryos, as well as embryonic growth retardation. In addition, the epithelium of zinc deficient embryos displayed a marked increase in surface microvilli, as well as the presence of blebbing. Transmission electron microscopy indicated extensive cell death in the neural epithelium which was apparently more severely damaged by zinc deficiency than were mesenchymal cells. Mitochondrial cristae were affected to a greater degree than any other membrane of the cell and cristael disintigration appeared to represent the principal cellular lesion preceding necrosis of neural cells and neural tube teratology.
Dietary zinc deficiency has been shown to be highly teratogenic in rats (l-6) and other animals (7,8), yet few studies have reported on the ultrastructural effects of zinc deficiency in the affected embryo. Macroscopically, embryos exposed to in utero zinc impoverishment often display serious neural tube defects (exencephalus, anencephalus, spina bifida) as well as abnormalities of most other organ systems (l-8). Biochemically, the mechanism underlying zinc-related teratogenesis is often attributed to a reduced rate of cell division, resulting from impaired synthesis of DNA in affected embryos (3, g-12), although Eckhert and Hurley (11) have suggested that other factors may be involved, as DNA synthesis is more affected by zinc deficiency in the head region of the embryo than in the rest of the body. Mention has recently been made to the role of zinc as a membrane stabilizer (13-15) and the possibility that membrane lipid peroxidation may contribute to embryonic dysmorphogenesis (16). Studies recently performed in this laboratory (17,18) have focussed attention on embryonic development in rats between days 8-11 of gestation, the period which coincides with closure of the neural tube and is therefore of greatest significance in relation to congenital abnormalities arising from impaired neurulation (19-21). Histological examination revealed extensive cell death in the neural tubes of embryos exposed to severe maternal zinc deprivation on days 8 and 9 of pregnancy, followed by overt neural tube teratology in II-day-old embryos (18). 0024-3025/88
$3.00
+
.OO
Copyright (c) 1988 Pergamon Journals Ltd.
890
Zinc Deficiency in the Rat Embryo
Vol. 42, No. 8, 1988
The present study was undertaken to extend these earlier findings and to examine the ultrastructural effects of zinc deficiency during this critical period of embryogenesis. Use was made of both scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and particular attention was paid to the effect of zinc depletion on the integrity of membranes and subcellular organelles. Materials and Methods Female Sprague-Dawley rats (approx 24Og) were housed individually from day 0 of gestation, and were fed a soybean-based diet (6), containing either <0.5 pg zinc/g diet (zinc deficient) or 100 ug zinc/g (zinc replete), which were fed respectively ad libitum or in amounts restricted to the average daily consumption of the zinc deficient group. On day 11 of gestation, maternal blood was collected for serum zinc analysis. Concurrently, embryos were removed and four embryos from each litter were randomly selected for EM examination, the remainder were used for protein determination (22). Embryos for EM studies were fixed (3% paraformaldehyde, 3% glutaraldehyde) for several hours and postfixed in 1% osmium tetroxide. Half were critically point dried (Balzers Union, FL 9496) and coated with carbon and gold/palladium for examination by SEM (ETEC Autoscan). The other half were embedded in Spurrs' resin (TAAB, Berkshire, UK), sectioned on an ultra-microtome (Porter-Blum MT 2-B) and stained with uranyl acetate/lead citrate for examination by TEM (Philips EM 300). RESULTS
Quantitative data Quantitative data in relation to the effect of zinc deficiency on development of the 11-day embryo is given in Table 1, and shows a strong association between low maternal serum zinc levels, reduced embryonic size and increased teratogenesis. Scannina electron microscony Low magnification SEM micrographs (Figure 1) confirmed the lack of rotation of the embryo and open neural tubes previously reported to be associated with zinc deficiency (6, 16-18). Higher magnification (Figure 2) revealed differences in the surface features of the cranial epithelium, with a far denser population of microvilli and increased blebbing evident in the zinc deficient embryos. Transmission electron microscopy Varying numbers of dead and dying cells were frequently seen in the epithelial cells of the neural tube (Figure 3), although very much less cellular necrosis was evident in the mesenchymal cells.
Vol. 42, No. 8, 1988
Zinc Deficiency in the Rat Embryo
891
TABLE 1 The Effect of Zinc Deficiency on Development of the ll-day Rat Embryo
Parameter*
Maternal serum zinc (pg/ml)+ Litter size+ Embryonic protein (pg/embryo)+ Somites+ Crown-rump length (mm)~ Total body length (rmnl Cardiac diameter (y) Head diameter (y> Head height (mm) Failure to rotate Open neural tube Tail defect No defect noted
Zinc replete
Zinc deficient
1.43f0.18 14.6 f0.7 246.2fg.J 24.Jf0.4
0.88*0.07** 14.4f0.4 120.5*6.5** 16.5*1.4** 1.58f0.09** 4.04*0.34** 0.82*0.04** 0.75f0.04** 0.72f0.04** 30% 53% 40% 40%
1.78kO.00
5.52f0.17 0.9g*o.o4 1.02iO.04 0.95*0.04 0%
20x++ 0%
80%
. * + ** ++
All data except maternal serum zinc, litter size and protein levels were obtained from SEM. Values represent means f standard error of lo-30 embryos in each treatment group. Pt0.005 (Analysis of Variance). Open neural tubes involved the posterior neuropore which only closes on day 11 and may not represent a developmental defect.
FIGURE 1 Scanning Electron Micrographs of ll-day Zinc Deficient Rat Embryos Showing (a) Failure to Rotate (b) Open Anterior Neuropore (Bar I 100 pm).
892
Zinc Deficiency in the Rat Embryo
Vol. 42, No. 8, 1988
Vol. 42, No. 8, 1988
Zinc Deficiency in the Rat Embryo
893
In addition to cell death, high magnification of the cellular organelles of a large number of randomly selected living cells in the neural tube and mesenchyme revealed a distortion of the mitochondria of the neural tube cells of zinc deficient embryos (Figures 4a and 4b). There is evidence of disruption of most cell membranes including those of the mitochondrion, nucleus, endoplasmic reticulum and cell boundary. Generally, it appeared that the cristael membrane of the mitochondria was damaged before other cell membranes became affected, although all membranes disintegrated to some extent as the cell became necrotic. Damage to cell membranes occurred more severely in the neural tube than in the mesenchymal or epithelial cells (Figures 4c and 4d), the latter cells also showing the presence of surface microvilli as seen under SEM.
FIGURE 3
Transmission Electron Micrograph of Neural Tube in an 11-day-old Zinc Deficient Rat Embryo (x 4,600). Discussion The present study on 11-day-old embryos confirms the general teratogenic consequences of gestational zinc deficiency previously reported in rats at the end of gestation (l-5, 12), and accords specifically with the earlier report by Record et al (16) concerning cellular necrosis in the neural tubes of 11-day-old zinc deficient embryos. Overall, substantially reduced growth and development was evident in the 11-day-old zinc deficient embryos as reflected by their lower levels of protein and fewer somites, as well as decreased embryonic dimensions. Mechanistically, several biochemical lesions may contribute to these effects, but it is likely that the diminished activities of many zinc metalloenzymes (16, 23-26) especially those involved in the processes of DNA replication and the transcription and translation of DNA into protein that are of significance. Teratological data obtained in the present study revealed severe dysmorphology of the ll-day-old zinc deficient embryos in relation to the three major defects investigated (failure to rotate, open neural tube, tail anomalies), which was consistent with the teratology reported by other workers on term fetuses. However in contrast to the
894
Zinc Deficiency in the Rat Embryo
Vol. 42, No. 8, 1988
2
e c
Vol. 42, No. 8, 1988
Zinc Deficiency in the Rat Embryo
895
latter studies, increased fetal death was not associated with zinc deficiency at this point of pregnancy, which may represent a stage in embryogenesis prior to the expression of many lethal, zinc-related terata. SEM of the surface features of ll-day-old embryos reported in the present study indicate a ring of microvilli around the border of the squamous epithelial cells, where the edges of the cells are thin in comparison with the cells themselves, and which therefore suggests a role for these microvilli in obtaining nutrients for the embryo from the amniotic fluid. In control embryos further microvilli were present in sparse numbers on the cell surface, but these numbers were increased dramatically in zinc deficient specimens. Although evidence to date is limited, and further studies are necessary on the microvillar density of the embryonic epithelium, it is tempting to speculate that the increase in microvilli associated with zinc deficiency represents an attempt by the embryonic cells to extract as much zinc as possible from the amniotic fluid. Some support for this notion may be gained from the report by others that zinc deficiency causes an increase in the villus density of the small intestine of rats (27) although a similar response by microvilli in this organ has not been observed (28). Increased blebbing on the surface of zinc deficient embryos reflects cells under stress, and may represent a method for the removal of large volumes of waste from cells that cannot be metabolized adequately by normal cellular processes in the affected cells. Variation in the size of the blebs may correspond to the level of excretory activity, or possibly even involve the extrusion of whole cells into the amniotic fluid. TEM of embryonic neural tissue shows significant disorganisation of several cellular membranes especially those of the mitochondrial cristae in the neural epithelium. The greater sensitivity of neuroblasts to zinc deficiency-related membrane damage compared with mesenchymal cells raises questions whether the injury arises from membrane lipid peroxidation, which is reduced in mesenchymal cells due to the improved availability of zinc from foetal blood in the primative blood vessels in that region. The abundance of dead or dying cells within the neural epithelium of the zinc deficient embryo appears to be the final result of the effects of zinc deficiency upon cellular membranes. Embryonic cell death associated with zinc deficiency has been reported recently (18) and is corroborated by the present observations. The cell death resulting from zinc deficiency in these experiments is not related to the cell death which normally occurs in the development of the central nervous system. In brains of control embryos dead and dying cells are rarely seen, except after the stage of neuronal migration which occurs at later stages of development, and after neuronal proliferation has ceased. It is not known whether the loss of cells caused by zinc deficiency results in changes in the numbers of cells involved in the normal programmed cell death. Taken together the present findings suggest a pattern of events in the zinc deficient embryo which includes an attempt by the embryo to increase zinc uptake from amniotic fluid by proliferation of epithelial microvilli. Continued deprivation of zinc results in membrane disintigration, especially in the mitochondrion and in neural cell death. Mitochondria of neural tube cells are apparently affected more than mitochondria of other body cells, which may underlie the greater vulnerability of the head region to zinc deficiency than occurs in the rest of the body. However, other reasons such as a zinc-dependent nerve growth factor (29) should also be considered, as more than one mechanism may be involved. Impaired proliferative activity of neural cells, and indeed of other somatic cells in the embryo, leads to retarded and distorted development, which in turn is reflected in embryonic
896
Zinc Deficiency in the Rat Embryo
vol. 42, No. 8, 1988
mortality and fetal teratogenesis. The novel observations reported in this paper concerning membrane disintegration in zinc deficient embryos raises the question whether this lesion represents the primary teratogenic mechanism of this condition, and if so to what extent is it related to membrane lipoperoxidation and to other antioxidant mechanisms. REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
L.S. HURLEY and HSWENERTON, Proc. Sot. Exp. Biol. Med., 123 692-696 (1966). L.S. HURLEY, J. GGWAN and H. SWENERTON, Teratology, 4 199-204 (1971). L.S. HURLEY and R.E. SHRADER, Neurobioloav of the Trace Metals Zinc and Conner, C.C. Pfeiffer, Ed. pp. 7-51, Academic Press, New York (1972). J. WARKANY and H.G. PETERING, Teratology, 2 319-334 (1972). J. WARKANY and H.G. PETERING, Am. J. Ment. Defic., u 645-653 (1973). I.R. RECORD, I.E. DREOSTI, R.S. TULSI and S.J. MANUEL, Teratology, 33 311-317 (1986). H. SWENERTON and L.S. HURLEY, J. Nutr., 110 575-583 (1980). R.M. HACKMAN and L.S. HURLEY, Teratology, 28 355-368 (1983). J.R. DUNCAN and L.S. HURLEY, Proc. Sot. Exp. Biol. Med. 159 39-43 (1978). I.E. RECORD and I.E. DREOSTI, Nutr. Rep. Int., 20 749-755 (1979). C.D. ECKHERT and L.S. HURLEY, J. Nutr., 107 855-861 (1977). I.E. DREOSTI, I.R. RECORD and S.J. MANUEL, Biol. Trace Element Res., 2 21-29 (1980). M. CHYAPIL, Life Sciences, 11 1041-1049 (1973). M. CHVAPIL, Med. Clin. North Am., 60 799-812 (1976). W.J. BETTGER and B.L. O'DELL, Life Sciences, s 1425-1438 (1981). I.E. DREOSTI, I.R. RECORD and S.J. MANUEL, Biol. Trace Element Res., 1 103-122 (1985). I.R. RECORD, I.E. DREOSTI and R.S. TULSI, Aust. J. Exp. Biol. Med. Sci., 63 65-71 (1985). I.R. RECORD, R.S. TULSI, I.E. DREOSTI and F.J.FRASER, Teratology, 12 397-405 (1985). G.D. SNELL and L.C. STEVENS, B~O~ORV of the Laboratory Mouse, E.L. Green, Ed. pp. 205-245, McGraw-Hill Book Co., New York (1966). N.A. BROWN and S. FABRO, Teratology, 26 65-78 (1981). K.L. MOORE (Ed) Before We are Born, W.B. Saunders Co. Philadelphia (1983). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem., 193 266-275 (1951). I.E. DREOSTI, P.C. GREY and P.J. WILKINS, S. Afr. Med. J., 4f! 1585-1588 (1972). J.A. DUERRE, K.M. FORD and H.H. SANDSTEAD, J, Nutr. 107 1082-1093 (1977). B.L. O'DELL, Fed. Proc., 42 2821-2822 (1984). A.D. GARNICA, W.Y. CHAN and O.M. RENNERT, Curr. Prob. Pediatr. 16 (1986). S. SOUTHON, J.M. GEE, C.E. BAYLISS, G.M. WYATT, N. HORN and I.T. JOHNSON, Br. J. Nutr., B 603-611 (1986). S.I. KOO and D.E. TURK, J. Nutr. 107 896-908 (1977). S.E. PATTISON and M.F. DUNN, Biochem. l4, 2733-2739 (1975).
Pergamon Journals
ZINC DEFICIENCY IN THE 11 DAY RAT EMBRYO: A SCANNING AND TRANSMISSION ELECTRON MICROSCOPE STUDY 2 A.J. Harding 192 , I.E. Dreosti 193 , and R.S. Tulsi .
1 2 3
CSIRO Division of Human Nutrition, Adelaide, Australia. Department of Anatomy and Histology, Adelaide Univeristy, Adelaide, Australia. To whom correspondence should be addressed. (Received in final form December 18, 1987)
Summary Zinc deficient rat embryos were obtained on the 11th day of pregnancy and examined by scanning and transmission electron microscopy. Scanning electron microscopy revealed an increase in the number of deformed embryos, as well as embryonic growth retardation. In addition, the epithelium of zinc deficient embryos displayed a marked increase in surface microvilli, as well as the presence of blebbing. Transmission electron microscopy indicated extensive cell death in the neural epithelium which was apparently more severely damaged by zinc deficiency than were mesenchymal cells. Mitochondrial cristae were affected to a greater degree than any other membrane of the cell and cristael disintigration appeared to represent the principal cellular lesion preceding necrosis of neural cells and neural tube teratology.
Dietary zinc deficiency has been shown to be highly teratogenic in rats (l-6) and other animals (7,8), yet few studies have reported on the ultrastructural effects of zinc deficiency in the affected embryo. Macroscopically, embryos exposed to in utero zinc impoverishment often display serious neural tube defects (exencephalus, anencephalus, spina bifida) as well as abnormalities of most other organ systems (l-8). Biochemically, the mechanism underlying zinc-related teratogenesis is often attributed to a reduced rate of cell division, resulting from impaired synthesis of DNA in affected embryos (3, g-12), although Eckhert and Hurley (11) have suggested that other factors may be involved, as DNA synthesis is more affected by zinc deficiency in the head region of the embryo than in the rest of the body. Mention has recently been made to the role of zinc as a membrane stabilizer (13-15) and the possibility that membrane lipid peroxidation may contribute to embryonic dysmorphogenesis (16). Studies recently performed in this laboratory (17,18) have focussed attention on embryonic development in rats between days 8-11 of gestation, the period which coincides with closure of the neural tube and is therefore of greatest significance in relation to congenital abnormalities arising from impaired neurulation (19-21). Histological examination revealed extensive cell death in the neural tubes of embryos exposed to severe maternal zinc deprivation on days 8 and 9 of pregnancy, followed by overt neural tube teratology in II-day-old embryos (18). 0024-3025/88
$3.00
+
.OO
Copyright (c) 1988 Pergamon Journals Ltd.
890
Zinc Deficiency in the Rat Embryo
Vol. 42, No. 8, 1988
The present study was undertaken to extend these earlier findings and to examine the ultrastructural effects of zinc deficiency during this critical period of embryogenesis. Use was made of both scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and particular attention was paid to the effect of zinc depletion on the integrity of membranes and subcellular organelles. Materials and Methods Female Sprague-Dawley rats (approx 24Og) were housed individually from day 0 of gestation, and were fed a soybean-based diet (6), containing either <0.5 pg zinc/g diet (zinc deficient) or 100 ug zinc/g (zinc replete), which were fed respectively ad libitum or in amounts restricted to the average daily consumption of the zinc deficient group. On day 11 of gestation, maternal blood was collected for serum zinc analysis. Concurrently, embryos were removed and four embryos from each litter were randomly selected for EM examination, the remainder were used for protein determination (22). Embryos for EM studies were fixed (3% paraformaldehyde, 3% glutaraldehyde) for several hours and postfixed in 1% osmium tetroxide. Half were critically point dried (Balzers Union, FL 9496) and coated with carbon and gold/palladium for examination by SEM (ETEC Autoscan). The other half were embedded in Spurrs' resin (TAAB, Berkshire, UK), sectioned on an ultra-microtome (Porter-Blum MT 2-B) and stained with uranyl acetate/lead citrate for examination by TEM (Philips EM 300). RESULTS
Quantitative data Quantitative data in relation to the effect of zinc deficiency on development of the 11-day embryo is given in Table 1, and shows a strong association between low maternal serum zinc levels, reduced embryonic size and increased teratogenesis. Scannina electron microscony Low magnification SEM micrographs (Figure 1) confirmed the lack of rotation of the embryo and open neural tubes previously reported to be associated with zinc deficiency (6, 16-18). Higher magnification (Figure 2) revealed differences in the surface features of the cranial epithelium, with a far denser population of microvilli and increased blebbing evident in the zinc deficient embryos. Transmission electron microscopy Varying numbers of dead and dying cells were frequently seen in the epithelial cells of the neural tube (Figure 3), although very much less cellular necrosis was evident in the mesenchymal cells.
Vol. 42, No. 8, 1988
Zinc Deficiency in the Rat Embryo
891
TABLE 1 The Effect of Zinc Deficiency on Development of the ll-day Rat Embryo
Parameter*
Maternal serum zinc (pg/ml)+ Litter size+ Embryonic protein (pg/embryo)+ Somites+ Crown-rump length (mm)~ Total body length (rmnl Cardiac diameter (y) Head diameter (y> Head height (mm) Failure to rotate Open neural tube Tail defect No defect noted
Zinc replete
Zinc deficient
1.43f0.18 14.6 f0.7 246.2fg.J 24.Jf0.4
0.88*0.07** 14.4f0.4 120.5*6.5** 16.5*1.4** 1.58f0.09** 4.04*0.34** 0.82*0.04** 0.75f0.04** 0.72f0.04** 30% 53% 40% 40%
1.78kO.00
5.52f0.17 0.9g*o.o4 1.02iO.04 0.95*0.04 0%
20x++ 0%
80%
. * + ** ++
All data except maternal serum zinc, litter size and protein levels were obtained from SEM. Values represent means f standard error of lo-30 embryos in each treatment group. Pt0.005 (Analysis of Variance). Open neural tubes involved the posterior neuropore which only closes on day 11 and may not represent a developmental defect.
FIGURE 1 Scanning Electron Micrographs of ll-day Zinc Deficient Rat Embryos Showing (a) Failure to Rotate (b) Open Anterior Neuropore (Bar I 100 pm).
892
Zinc Deficiency in the Rat Embryo
Vol. 42, No. 8, 1988
Vol. 42, No. 8, 1988
Zinc Deficiency in the Rat Embryo
893
In addition to cell death, high magnification of the cellular organelles of a large number of randomly selected living cells in the neural tube and mesenchyme revealed a distortion of the mitochondria of the neural tube cells of zinc deficient embryos (Figures 4a and 4b). There is evidence of disruption of most cell membranes including those of the mitochondrion, nucleus, endoplasmic reticulum and cell boundary. Generally, it appeared that the cristael membrane of the mitochondria was damaged before other cell membranes became affected, although all membranes disintegrated to some extent as the cell became necrotic. Damage to cell membranes occurred more severely in the neural tube than in the mesenchymal or epithelial cells (Figures 4c and 4d), the latter cells also showing the presence of surface microvilli as seen under SEM.
FIGURE 3
Transmission Electron Micrograph of Neural Tube in an 11-day-old Zinc Deficient Rat Embryo (x 4,600). Discussion The present study on 11-day-old embryos confirms the general teratogenic consequences of gestational zinc deficiency previously reported in rats at the end of gestation (l-5, 12), and accords specifically with the earlier report by Record et al (16) concerning cellular necrosis in the neural tubes of 11-day-old zinc deficient embryos. Overall, substantially reduced growth and development was evident in the 11-day-old zinc deficient embryos as reflected by their lower levels of protein and fewer somites, as well as decreased embryonic dimensions. Mechanistically, several biochemical lesions may contribute to these effects, but it is likely that the diminished activities of many zinc metalloenzymes (16, 23-26) especially those involved in the processes of DNA replication and the transcription and translation of DNA into protein that are of significance. Teratological data obtained in the present study revealed severe dysmorphology of the ll-day-old zinc deficient embryos in relation to the three major defects investigated (failure to rotate, open neural tube, tail anomalies), which was consistent with the teratology reported by other workers on term fetuses. However in contrast to the
894
Zinc Deficiency in the Rat Embryo
Vol. 42, No. 8, 1988
2
e c
Vol. 42, No. 8, 1988
Zinc Deficiency in the Rat Embryo
895
latter studies, increased fetal death was not associated with zinc deficiency at this point of pregnancy, which may represent a stage in embryogenesis prior to the expression of many lethal, zinc-related terata. SEM of the surface features of ll-day-old embryos reported in the present study indicate a ring of microvilli around the border of the squamous epithelial cells, where the edges of the cells are thin in comparison with the cells themselves, and which therefore suggests a role for these microvilli in obtaining nutrients for the embryo from the amniotic fluid. In control embryos further microvilli were present in sparse numbers on the cell surface, but these numbers were increased dramatically in zinc deficient specimens. Although evidence to date is limited, and further studies are necessary on the microvillar density of the embryonic epithelium, it is tempting to speculate that the increase in microvilli associated with zinc deficiency represents an attempt by the embryonic cells to extract as much zinc as possible from the amniotic fluid. Some support for this notion may be gained from the report by others that zinc deficiency causes an increase in the villus density of the small intestine of rats (27) although a similar response by microvilli in this organ has not been observed (28). Increased blebbing on the surface of zinc deficient embryos reflects cells under stress, and may represent a method for the removal of large volumes of waste from cells that cannot be metabolized adequately by normal cellular processes in the affected cells. Variation in the size of the blebs may correspond to the level of excretory activity, or possibly even involve the extrusion of whole cells into the amniotic fluid. TEM of embryonic neural tissue shows significant disorganisation of several cellular membranes especially those of the mitochondrial cristae in the neural epithelium. The greater sensitivity of neuroblasts to zinc deficiency-related membrane damage compared with mesenchymal cells raises questions whether the injury arises from membrane lipid peroxidation, which is reduced in mesenchymal cells due to the improved availability of zinc from foetal blood in the primative blood vessels in that region. The abundance of dead or dying cells within the neural epithelium of the zinc deficient embryo appears to be the final result of the effects of zinc deficiency upon cellular membranes. Embryonic cell death associated with zinc deficiency has been reported recently (18) and is corroborated by the present observations. The cell death resulting from zinc deficiency in these experiments is not related to the cell death which normally occurs in the development of the central nervous system. In brains of control embryos dead and dying cells are rarely seen, except after the stage of neuronal migration which occurs at later stages of development, and after neuronal proliferation has ceased. It is not known whether the loss of cells caused by zinc deficiency results in changes in the numbers of cells involved in the normal programmed cell death. Taken together the present findings suggest a pattern of events in the zinc deficient embryo which includes an attempt by the embryo to increase zinc uptake from amniotic fluid by proliferation of epithelial microvilli. Continued deprivation of zinc results in membrane disintigration, especially in the mitochondrion and in neural cell death. Mitochondria of neural tube cells are apparently affected more than mitochondria of other body cells, which may underlie the greater vulnerability of the head region to zinc deficiency than occurs in the rest of the body. However, other reasons such as a zinc-dependent nerve growth factor (29) should also be considered, as more than one mechanism may be involved. Impaired proliferative activity of neural cells, and indeed of other somatic cells in the embryo, leads to retarded and distorted development, which in turn is reflected in embryonic
896
Zinc Deficiency in the Rat Embryo
vol. 42, No. 8, 1988
mortality and fetal teratogenesis. The novel observations reported in this paper concerning membrane disintegration in zinc deficient embryos raises the question whether this lesion represents the primary teratogenic mechanism of this condition, and if so to what extent is it related to membrane lipoperoxidation and to other antioxidant mechanisms. REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
L.S. HURLEY and HSWENERTON, Proc. Sot. Exp. Biol. Med., 123 692-696 (1966). L.S. HURLEY, J. GGWAN and H. SWENERTON, Teratology, 4 199-204 (1971). L.S. HURLEY and R.E. SHRADER, Neurobioloav of the Trace Metals Zinc and Conner, C.C. Pfeiffer, Ed. pp. 7-51, Academic Press, New York (1972). J. WARKANY and H.G. PETERING, Teratology, 2 319-334 (1972). J. WARKANY and H.G. PETERING, Am. J. Ment. Defic., u 645-653 (1973). I.R. RECORD, I.E. DREOSTI, R.S. TULSI and S.J. MANUEL, Teratology, 33 311-317 (1986). H. SWENERTON and L.S. HURLEY, J. Nutr., 110 575-583 (1980). R.M. HACKMAN and L.S. HURLEY, Teratology, 28 355-368 (1983). J.R. DUNCAN and L.S. HURLEY, Proc. Sot. Exp. Biol. Med. 159 39-43 (1978). I.E. RECORD and I.E. DREOSTI, Nutr. Rep. Int., 20 749-755 (1979). C.D. ECKHERT and L.S. HURLEY, J. Nutr., 107 855-861 (1977). I.E. DREOSTI, I.R. RECORD and S.J. MANUEL, Biol. Trace Element Res., 2 21-29 (1980). M. CHYAPIL, Life Sciences, 11 1041-1049 (1973). M. CHVAPIL, Med. Clin. North Am., 60 799-812 (1976). W.J. BETTGER and B.L. O'DELL, Life Sciences, s 1425-1438 (1981). I.E. DREOSTI, I.R. RECORD and S.J. MANUEL, Biol. Trace Element Res., 1 103-122 (1985). I.R. RECORD, I.E. DREOSTI and R.S. TULSI, Aust. J. Exp. Biol. Med. Sci., 63 65-71 (1985). I.R. RECORD, R.S. TULSI, I.E. DREOSTI and F.J.FRASER, Teratology, 12 397-405 (1985). G.D. SNELL and L.C. STEVENS, B~O~ORV of the Laboratory Mouse, E.L. Green, Ed. pp. 205-245, McGraw-Hill Book Co., New York (1966). N.A. BROWN and S. FABRO, Teratology, 26 65-78 (1981). K.L. MOORE (Ed) Before We are Born, W.B. Saunders Co. Philadelphia (1983). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem., 193 266-275 (1951). I.E. DREOSTI, P.C. GREY and P.J. WILKINS, S. Afr. Med. J., 4f! 1585-1588 (1972). J.A. DUERRE, K.M. FORD and H.H. SANDSTEAD, J, Nutr. 107 1082-1093 (1977). B.L. O'DELL, Fed. Proc., 42 2821-2822 (1984). A.D. GARNICA, W.Y. CHAN and O.M. RENNERT, Curr. Prob. Pediatr. 16 (1986). S. SOUTHON, J.M. GEE, C.E. BAYLISS, G.M. WYATT, N. HORN and I.T. JOHNSON, Br. J. Nutr., B 603-611 (1986). S.I. KOO and D.E. TURK, J. Nutr. 107 896-908 (1977). S.E. PATTISON and M.F. DUNN, Biochem. l4, 2733-2739 (1975).