CONGENITAL MALFORMATIONS OF THE NERVOUS SYSTEM IN ZINC-DEFICIENT RATS*

Lucille S. Hurley and Ruth E. Shrader,     Department of Nutrition, University of California, Davis, California

Publisher Summary

This chapter discusses congenital malformations of the nervous system in zinc-deficient rats. Zinc deficiency appears to have a most marked effect on growing or proliferating tissues. Young weanling rats in a rapid stage of growth are severely affected by zinc deficiency and show extreme retardation or cessation of growth. Similarly, there is a complete arrest of ovarian maturation in the female and of spermatogenesis in the male. The deleterious effect of zinc deficiency on rapidly growing tissues is also very pronounced in the embryo. In an experiment described in the chapter, zinc deficiency in pregnant rats resulted in a high rate of embryonic death, severe intrauterine growth retardation, and a high incidence of congenital malformations affecting every organ system. In the experiment, rat embryos from zinc-deficient females showed a reduced uptake of tritiated thymidine when compared with controls, as measured by liquid scintillation and autoradiography.

I. Introduction 7

A. Effects of Zinc Deficiency on Development

B. Movement of Zinc in the Maternal-Fetal Organism

II. Gross Congenital Malformations in Zinc-Deficient Rats

A. General Methods Used

B. Effects of Short-Term and Transitory Zinc Deficiency

III. Histopathology of Nervous System Malformations in Zinc Deficiency

A. Material

B. Gross External Morphology of Fetuses Studied Histologically

C. Pattern of Nervous System Malformations in Zinc-Deficient Fetal Rats

D. Morphogenesis of the Fetal Rat Brain

IV. Concluding Remarks

    References

I. Introduction

Zinc is an essential nutrient for both animals and plants. It is found in animal tissues in very small (trace) amounts, and must be available from the diet or from other environmental sources in small but important amounts in order to maintain life, growth, and reproduction. The metabolism of zinc, effects of its deficiency, and its biochemical functions have been reviewed (Underwood, 1962; Vallee, 1962; Prasad, 1966; Prasad, 1969; Mills, 1970).

A. EFFECTS OF ZINC DEFICIENCY ON DEVELOPMENT

Zinc deficiency appears to have a most marked effect on growing or proliferating tissues (Hurley, 1969). Thus, young weanling rats in a rapid stage of growth are severely affected by zinc deficiency, and show extreme retardation or cessation of growth (Swenerton and Hurley, 1968). Similarly, there is a complete arrest of ovarian maturation in the female (Swenerton and Hurley, 1968); and of spermatogenesis in the male (Swenerton and Hurley, 1968; Diamond et al., 1971). In fact, the effect of zinc deficiency on the testis of the weanling rat was so rapid that microscopic lesions were apparent in this organ within one week after the beginning of a zinc deficiency regime (Diamond et al., 1971).

The deleterious effect of zinc deficiency on rapidly growing tissues is also very pronounced in the embryo. When hens were fed a zinc-deficient diet, chicks hatched from their eggs were weak and died within 4 days (Turk et al., 1959). With more-severely-deficient hens, gross malformations were observed in chick embryos including skeletal defects, brain abnormalities, and herniation of viscera (Blamberg et al., 1960; Keinholz et al., 1961). Zinc deficiency in pregnant rats resulted in a high rate of embryonic death, severe intrauterine growth retardation, and a high incidence of congenital malformations affecting every organ system (Hurley and Swenerton, 1966; Hurley et al., 1971).

Rapidly developing tissues such as those of the fetus and the testis of young males may be particularly sensitive to zinc inadequacy because of a requirement for zinc at the fundamental level of nucleic acid metabolism. Rat embryos from zinc-deficient females showed a reduced uptake of tritiated thymidine when compared with controls, as measured by liquid scintillation and autoradiography. Scintillation counting of whole embryos showed that incorporation of labeled thymidine into the embryos of zinc-deficient rats was significantly lower than in controls, whether based on the count per whole embryo or per unit weight. Audoradiographs showed that cells of zinc-deficient embryo tissues contained less tritiated thymidine than those of controls. In both groups the highest concentration of label was found in the outer half of the primitive ependymal zone of the cerebral vesicles, the undifferentiated mesenchyme, embryonic blood, and liver cells. Heart and somatic muscle, and lining cells of the primitive gut, trachea, and bronchi were less-heavily labeled in both deficient and control tissues (Swenerton et al., 1969).

Requirement of zinc ion for DNA synthesis has also been demonstrated in liver perfused with ethylenediaminetetraacetate (EDTA) in partially hepatectomized rats (Fujioka and Lieberman, 1964). Dietary zinc deficiency also decreased the incorporation of thymidine into nuclear DNA of liver cells in young growing rats (Sandstead and Rinaldi, 1969).

B. MOVEMENT OF ZINC IN THE MATERNAL–FETAL ORGANISM

The extremely rapid effect of zinc deficiency arises from the need for a constant extraneous source of zinc in order to maintain plasma levels of the element (Dreosti et al., 1968). When rats were given a zinc-deficient diet at the beginning of pregnancy, plasma zinc concentration dropped rapidly. After only 24 hours of the zinc deficiency regime, plasma zinc fell by approximately 38% (from 96 to 60 μg/100 ml) and a plateau was reached at about 30 μg/100 ml after 14 days. Similar observations were made with weanling rats (Dreosti et al., 1968).

The rapid effect of the zinc-deficiency regime appeared to be brought about by lack of mobilization of zinc from maternal stores for the benefit of the fetus. Although under these conditions maternal plasma zinc concentrations were not decreased by pregnancy (Hurley and Swenerton, 1971). Thus there appears to be little homeostatic control of plasma zinc levels; the pregnant rat cannot mobilize zinc from the relatively large amounts in her skeleton, or from other body deposits, in order to supply the needs of her developing fetuses. Thus the maternal—fetal organism cannot adjust to even a relatively short period of dietary restriction of zinc.

II. Gross Congenital Malformations in Zinc-Deficient Rats

The rapid growth and severe effects of zinc defiency on the embryo can be readily demonstrated by studies of short-term and transitory zinc deficiency during pregnancy. The gross congenital malformations occurring under such conditions will be reviewed in this paper. In addition, we will present some original previously unpublished histological findings on abnormal development of the nervous system resulting from this deficiency. The histopathology of certain tissues in zinc-deficient fetuses has been described (Diamond and Hurley, 1970).

A. GENERAL METHODS USED

In all work with zinc deficiency not only it is important to use a diet which is low in zinc, but it is equally important to prevent availability of zinc from sources other than the diet. This is neccessary in order that the amount of zinc ingested by the animals be known and a severe zinc deficiency state may be induced.

The basic procedures of diet composition, diet preparation, and environmental control that were developed in this laboratory have been followed in all of the work that is summarized in this review. The ration used had the following percentage composition: isolated soybean protein,* 30.0; sucrose, 57.3; corn oil, 8.0; salts,† 4.0; and DL-methionine, 0.7. The zinc content of the soybean protein was reduced by treatment with EDTA. Crystalline vitamins were given separately.‡ The basal zinc-free ration used in the various experiments contained 0.20-0.60 ± 0.05 ppm of zinc as determined by atomic absorption spectroscopy. Zinc-supplemented control animals received the same diet except that zinc carbonate was added to the salt mix, providing a total content in the diet of 100 ppm of zinc.

Stainless steel cages and racks were used in all experiments, and all rats were housed individually, except for the brief periods when a male was introduced into the cage for breeding purposes. Deionized water was given in Pyrex bottles with vinyl plastic stoppers and stainless steel...