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HORMONES AND VITAMINS IN PRENATAL LIFE Hormonal and vitamin imbalances which cause malaise or even pass unnoticed in post-natal life, may have a disastrous effect on the developing foetus. The rapid rate of growth of embryonic cells, which far surpasses that of most tumour cells, is the basic factor which determines the exaggerated foetal response to injurious stimuli. Interference with growth or metabolism of developing cells at an early stage is reflected ultimately in alterations at the stage of differentiation, with resulting malformations or impaired function of developing organs and tissues. The embryonic cell has much in common with the malignant cell, including high mitotic rate, and lack of differentiation in the early stages. It is therefore not surprising that most of the agents discussed in the previous chapter as being responsible for malignant transformation of cells are equally capable of causing dysmorphogenesis in the developing foetus. Early foetal development depends to a certain extent on hormones supplied by the maternal endocrine organs and the placenta, and on vitamins and other food factors derived from the maternal blood supply. Eventually, however, each foetal endocrine organ grows, differentiates and matures, and in those animals which are born at a relatively well developed state, produce their own hormones, many of which are essential for further normal development of their receptor organs and tissues. As each organ develops, it influences the growth and maturation of other organs, and eventually the functional relationships between endocrine glands characteristic of post natal life become established. Thus, normally, the pituitary-adrenal and the pituitary-gonadal axes are fully functional before birth. Receptor organs respond to hormonal stimulation when they acquire sufficient maturity to do so. The earliest growth and differentiation of endocrine glands is independent of stimulation by other foetal glands, but prenatal maturation requires the presence of a fully functional hormonal network. The difficulties in working with foetal tissues are so great that we have little direct knowledge of the biosynthetic pathways by which the foetal endocrine glands synthesise their hormones, but there is no reason to believe that they are very different from the modes of hormonal synthesis in post-natal glands. Assuming this metabolic similarity, one must accept that vitamins are as essential for the synthesis and operation of prenatal hormones as they are for post-natal hormones. Such an assumption is supported by the abnormalities of endocrine development and function found in animals partly or completely deprived of vitamins during the course of foetal life. The absence of the vitamin-containing coenzymes required for biochemical sequences in foetal organogenesis and metabolism has far-reaching effect. Many of the experimentally induced vitamin deficiencies cause malformations identical with those reputed to be hereditary in origin. As a rule, the demands of the mother for vitamins take priority over foetal demands, so that in many cases there many be no sign of maternal deficiency in essential food factors, while a the same time the foetus may be suffering marked deficiency. This means that a marginal maternal deficiency, which is very difficult to detect, may have grave consequences for the foetus. Not all vitamin deficiencies cause recognisable defects in foetal development. Some cause foetal death, others provoke malformation. Many simply cause retarded growth, by interfering with foetal metabolic processes in a minor degree. Individual requirements for vitamins vary over a wide range, so that deficiency which proves teratogenic in one strain of animal, or in one individual, may not necessarily cause developmental defects in another strain, or individual. It seems therefore that genetic factors are important in determining susceptibility to vitamin deficiencies. The subject of foetal nutrition is a very complicated matter, since each step in morphogenesis, as for example the formation of the cardiovascular system or the central nervous system, has its own individual requirements, both qualitative and quantitative, which must be satisfied if ontogenesis is to proceed normally. Knowledge of such requirements can only be attained by observing the results of the relevant deficiencies in experimental animals and in the very few cases in man in which a single conditioned deficiency results from therapeutic treatment. Such a condition arises for example when aminopterin, a folic acid analogue, is used to induce therapeutic abortion, and fails to achieve this object. The conditioned deficiency of folic acid which is thus induced gives rise to multiple congenital defects. There is a marked resemblance between the pattern of congenital defects produced by hormonal deficiency, and that produced by vitamin deficiencies. The same pattern is induced too by chemical and viral teratogens and by irradiation, and it is the specific period of pregnancy during which the noxious stimulus acts which determines the pathological effect, rather than the teratogen itself. The whole wide range of defects in human morphogenesis can be duplicated in most cases in experimental animals by dietary manipulation. These defects may be grouped into:-
A further category of congenital defect could be included, to cover overgrowth of tissues, but these might be more accurately classified as congenital tumours. Carter (1968) lists the most common malformations of new born children in England and Wales as anencephaly, spina bifida, Down's syndrome, pyloric stenosis, cleft lip (with or without cleft palate), talipes equinovarus, congenital hip dislocation and congenital heart malformation. All these conditions, with the exception of Down's syndrome (mongolism) he attributes to the effect of a polygeneic genetic predisposition, triggered off by some unknown intrauterine environmental factor. 'If' as Carter says ' the additional factors are, as they may well prove to be, nutritional, then it is reasonable to hope that we can protect the mother, and through her the foetus.' It is known of course that virus infections, irradiation and chemical teratogens such as thalidomide cause a variety of congenital defects, but such cases form a small fraction of the total of foetal abnormalities and it seems that much more could be done in the field of preventive medicine to cut down the large numbers of preventable congenital defects. In human foetal development, most abnormalities are established by the eight to the tenth week of gestation. This means of course that the most important period for nutritional care occurs in the few weeks before and immediately after conception. This is not usually the time when diet is considered important. Developmental defects following maternal rubella infection, and treatment with thalidomide have spot-lighted dramatically the vulnerability of the foetus at an early stage of development. Nevertheless, most of our advances in knowledge of the interaction of the foetus with its environment must come from work with experimental animals. Although there are obvious dangers in extrapolation of the results gained from animal experiments, there is much to be learned from a comparative study. The animals most in use are rabbits, rats and mice, of course the developing hen's egg provides the most easily accessible experimental model. It should be realised that there is much variation in the state of maturity of animals at the end of their period of gestation. The newly born rat and mouse are at a retarded state of maturity when compared with ruminants, some of which are able to rise to their feet and run with the herd within half an hour of birth. Comparison, then with stages of human development requires a detailed knowledge of the timetable of morphogenetic events in the animal concerned, as compared with that of man. A valuable contribution in this field has been provided by Otis and Brent (1954) who have drawn up a graphic comparison of the times of appearance of 147 recognisable stages in development in mice and men. There is close comparison between the times of appearance of structures in the first seventeen days of mouse foetal life, and the first fifteen weeks of human foetal life. The total gestation period in the mouse is 19 days, therefore in comparison with man, the newly born mouse is distinctly underdeveloped. The rat has a gestation period of 22 days. At the end of this time the rat thyroid follicles are just starting to show a small amount of colloid, whereas in man, a similar stage is reached at 8-10 weeks. In the human foetus the adrenal medulla begins to function at 12 weeks and the adrenal cortex shows signs of secretory ability at or before 9 weeks. In the rat, migration of adrenal medullary cells into the primitive adrenal cortex does not begin until the 16th day of foetal life. Even at birth, cortical tissue is still undifferentiated. In man, the pancreatic islets become differentiated at 12-14 weeks compared with differentiation at the 19th or 20th day in the rat, whose beta-cells start producing insulin on the 22nd day. The alpha cells do not differentiate until after birth. The human anterior pituitary is well differentiated by the 12th week, that is he end of the first trimester, a stage of development only reached by the rat towards the end of the gestation period. Similarly, sexual differentiation starts at 6 or 7 weeks in man, and at 13 or 14 days in the rat. Keeping these marked differences in stages of maturity constantly in mind, it is possible to draw some comparisons between anomalies in development in human and animal foetuses caused by various factors acting at comparable critical periods in organogenesis. Such critical periods may occur just before the rudiments of an organ are detectable, or when the cells in the developing organ are most actively dividing. The formation of the anlagen (primitive masses of cells forming the rudiments of organs) and successive steps in differentiation of human endocrine glands related to length and age of embryos are described by Tonutti and Fetzer (1956) as also are the postnatal changes in maturation of endocrine glands up to the age of puberty. This work forms a useful reference for the minuter details of endocrine development. As we have seen, the whole spectrum of human foetal abnormalities can be simulated experimentally in properly chosen animals, by dietary adjustments, at strictly defined periods of gestational time. This provides a valuable means of investigating the faults in the basic biochemical mechanisms which underlie the pathological phenomena so produced. Foetal anomalies may arise as a result of faulty implantation of the ovum, or as a result of placental disease, but we are concerned here only with those defects which arise as a result of faulty hormonal balance or dietary deficiency. Deformities may arise directly in the cells of the deformed structure. Or they may be caused indirectly by faulty nutrition arising from insufficient maternal blood supply. Or again the foetal cardiovascular or hepatic output may be at fault, or there may be lack of control by the foetal endocrine system. Careful control of the experimental model is essential. It is necessary to initiate a borderline deficiency or imbalance, since excessive interference with maternal nutrition will result in sterility, or death and resorption of the foetus. The same situation applies in the field of human nutrition where both high infant mortality and high incidence of foetal anomalies are seen in the underdeveloped countries, following maternal malnutrition. Pathogenesis of single vitamin deficiencies Vitamin A. The list of congenital abnormalities produced in experimental animals by maternal A deficiency is a long one. The details vary with the species of animal used, the intensity, and in particular the timing of the deficiency in pregnancy. The large number of malformations indicate a primary effect on the process of organogenesis. Piglets are born without eyes or with microphthalmos. Calves become blind from constriction of the optic nerve by relative overgrowth of osseous tissue. Rabbits develop hydrocephalus. It is the rat however, which shows the widest range of foetal abnormalities, including malformations of the eye, cardiovascular anomalies, resembling those seen in man, urogenital anomalies, diaphragmatic hernia, hypospadias and cryptorchidism. The last anomaly, failure of the testes to descend, is said to be due to the failure of the testicular Leydig cell to produce sufficient hormone to induce the descent. In view of the necessity of vitamin A for the synthesis of corticosteroids and testosterone, this explanation would appear to satisfactory. Excess vitamin A given to pregnant females is also teratogenic. Deformities include cranial anomalies, cleft palate, harelip and eye defects, hydrocephalus, spina bifida and xencephalos. The teratogenic action of vitamin A excess in rats is potentiated by cortisone, although cortisone is not by itself teratogenic. The combined action is difficult to explain, as cortisone normally stabilizes the membranes which are said to be damaged by vitamin A excess. As has already been mentioned excess vitamin A damages lipoprotein membranes. The organs distorted in vitamin A excess are derived from the neural tube, the lens primordia, and the oral cavity, and it is presumed that embryopathic changes are the result of direct attack by vitamin A on these ectodermal structures, with alteration of the molecular arrangement of their cell membranes. Protection against the brain anomalies is given by the injection of protamine zinc insulin between the ninth and twelfth days of pregnancy. Vitamin A is said to be able to pass through the human placenta, although carotene is retained. Premature babies are known to be deficient in vitamin A, perhaps because they have not had time to build up sufficient stores of the vitamin for the stressful first few days of life. Their diet should be supplemented with the preformed vitamin until their bodily stores become adequate. The children of women with diabetes mellitus have a higher incidence of malformations than the children of non-diabetic women. Microcephaly, hydrocephalus, cardiac defects and cleft palate are common. Similarly the offspring of diabetic rabbits show gross deformities of the brain. It is reasonable to suggest that vitamin A deficiency is the basic cause, or one of the basic causes of such changes, since diabetic subjects are unable to convert carotene to vitamin A. If this fact is not recognised, and the subject given preformed vitamin A then such abnormalities might be expected in the children of a certain proportion of women at risk. It is essential too that at each level of embryonic development, the foetal blood glucose level should be in correct co-ordination with requirements; marked deviations from normal result in foetal malformation. It was thought at one time that mongolism was the result of defective transfer of vitamin A from the mother to the foetus. Although mongolism is now thought to be a hereditary defect associated with trisomy in chromosome 21, the two theories are not contradictory, in view of recent investigations in the induction of chromosome anomalies by conditioned vitamin deficiency (vide infra). The vitamin B complex. The necessity of members of the vitamin B complex for correct operation of the glycolytic pathway, the citric acid cycle and the electron transfer mechanism is emphasised by the marked disturbance of foetal metabolism caused by their absence. Often, a deficiency of a single metabolite lasting no more than a day can be shown to produce foetal abnormalities in rats. It will be recalled of course that these water soluble vitamins are not stored in the animal body. This means that animals are more readily depleted of B vitamins than they are of the fat soluble A vitamin. Foetal avitaminosis A is not readily achieved unless the dam is deprived of the vitamin from the time of weaning. As might be expected, riboflavin deficiency causes, in the foetal liver, reduced oxygen consumption and reduced activity of such enzymes as succinic dehydrogenase and cytochrome oxidase. Severe deficiency of course causes sterility in rats, but borderline deficiency may allow the development of small misshapen foetuses, which often die in utero and become resorbed. Although there are differences in susceptibility to the deficiency in different strains, the general syndrome is one of skeletal malformation and blood disorder. Micromelia (abnormally small limbs), short mandibles, cleft palate, and syndactylism with fused cartilaginous anlagen are all seen; foetuses are oedematous and anaemic. Degeneration of Wolffian bodies can often be detected. Deficiency induced by the antivitamin has been known to cause anophthalmia and microphthalmia. Thiamine deficiency is responsible for anoestrus with sterility, or relative infertility in rats, depending on the degree of deprivation. Marginally deficient rats may become pregnant and give birth to undersized progeny, but the perinatal mortality rate is high. Thiamine is apparently required most urgently in the late stages of pregnancy, and foetal morphological anomalies are therefore not associated with deficiency of this vitamin, either in experimental animals or in human beri-beri patients. Embryopathy in thiamine deficient young is probably associated with pituitary or ovarian malfunctions, since, as Nelson and Evans (1956) have shown, foetal death and resorption can be prevented by daily injections of oestrone and progesterone during pregnancy. Nicotinic acid. There are apparently no foetal abnormalities in man associated with pellagra, but in experimental animals, development abnormalities can be induced by short term use of nicotinic acid antagonists. Pinsky and Fraser (1960) by giving 6-aminonicotinamide to mice on the ninth day of gestation were able to produce cleft lip and palate, and hind limb defects. The inhibition of nicotinamide adenine dinucleotide dependent reactions was suggested as the cause of the anomalies. The timing was critical. Inhibition on the tenth day failed to cause the anomalies. Even as short an inhibition as two hours (determined by giving nicotinamide two hours after the antagonist) was sufficient to cause lasting damage to the foetus. Similar malformations may be induced in foetal rabbits by the use of a nicotinic acid antagonist at a critical period in morphogenesis. Biotin. Low levels of dietary biotin are not apparently teratogenic in the rat, but the progeny of deficient pregnant females may be resorbed in utero or fail to survive more than a few days of post-natal life. Degenerative changes may be found in the heart and blood vessels, and in the liver. Pantothenic acid deficiency gives rise to foetal abnormalities mainly affecting the nervous system. The timing of the deficiency during gestation is of critical importance. The period of greatest need appears to be just before birth, in animals born mature. The need for coenzyme A, a pantothenic acid containing cofactor, for phospholipid synthesis, may be one of the basic factors in determining integrity of the nervous system. The vulnerable periods for myelination in the brain in the dog, man, rat and pig have been discussed by Davison and Dobbing (1966). These authors state that myelination starts at different times in various areas of the nervous system, and the onset of myelination varies for each species. Myelin of course has a high content of phospholipid and defective synthesis may be expected to impair function to a certain extend prenatally, as it does post-natally in the demyelinating disease which lack of pantothenic acid causes. Folic acid is required for nucleic acid synthesis and as might be expected deficiency during pregnancy interferes with the normal development of organs and tissues. The effects produced in rats are similar to those induced by giving purine analogues (Kury et al., 1968), which likewise are antagonists of nucleic acid synthesis. Mercaptopurine is an example of the latter. This drug has been used extensively for the treatment of acute leukaemia in man. If mercaptopurine, or aminopterin (a folic acid antagonist) is given to pregnant rats, the foetuses develop with cleft palate and harelip, deformed limbs, and malformations of the heart, diaphragm, urogenital system and adrenals. The folic acid antagonist is known to cause the formation of spina bifida and to affect the development of the eye, and the closure of the body wall. There is a very broad spectrum of foetal abnormalities associated with folic acid deficiency. The incidence, and type of change vary with the degree and the duration of the deficiency. Even as short a period as 48 hours' deficiency causes a high incidence of abnormalities, the nature of which is determined by the gestational stage during which the deficiency is established (Nelson et al., 1955). Foetal death and resorption in folic acid deficient rats are not prevented by the administration of the sex hormones. It is interesting that the human foetus is able to concentrate folic acid from the maternal circulation. Foetal requirements for the vitamin are presumably much higher than maternal requirements, in order to keep pace with the tremendous mitotic activity occasioned by intra-uterine growth. Variations in the maternal level of vitamin B12 during pregnancy do not seem to affect the embryo. Vitamin B12 is one of the water soluble vitamins which the foetus is able to accumulate and retain at a higher level than that found in the maternal circulation. The production of skeletal deformities in the young born to rachitic females is associated with maternal deficiency of vitamin D. Prolonged severe E deficiency in rats causes sterility; in less severe deficiency implantation may take place, but the foetuses usually become resorbed and there is regression of the corpora lutea. Marginally deficient rats given alpha-tocopherol supplement before the ninth day of gestation are able to produce normal young. If the treatment is delayed until the twelfth day, then the young animal may develop exencephaly, hydrocephalus, syndactyly and an oedematous condition of the tissues. Abnormalities in the E deficient young rat are thought to be due to degenerative changes in, and haemorrhages from, the placental vessels. The resulting drop in blood supply causes foetal malnutrition and asphyxiation. Using the developing chick embryo as an experimental model Adamstone (1931) was able to demonstrate in E deficiency the presence of a ring of intensive cell proliferation in the blastoderm. The vitelline blood vessels were choked by the development of this lethal ring, and became degenerate. Many chick embryos suffered massive haemorrhage into the exocoele, usually from a ruptured cardiac atrium. The occurrence of abnormalities in marginally deficient rats is mitigated to a certain extent by oestrone or progesterone injections, but in the more severe deficiency characterised by foetal resorption, hormones appear to have no curative effect. In essential fatty acid deficiency embryos fail to maintain their normal rate of growth, and there is a low rate of neonatal survival. The blood vessels appear to undergo degenerative change and there may be haemorrhages into the tissues of the limbs and tail in the rat. Turning now from the problems created by failure to provide exogenous supplies of essential food factors to the developing foetus, we must next consider the problems arising from failure of the foetal glands to develop the harmonious relationships which are essential for normal growth. It is known that abnormal foetal hormone production is responsible for both metabolic disorders and gross morphological errors in development. Illustrative of these are the vascular derangements caused by excess adrenalin, and the defective closure of the palate caused by excess ACTH. Defects in the receptor tissues are the cause of dysharmony in the endocrine system. In studying the principles which govern hormonal action, we have already seen that:
These two principles apply in prenatal life, just as they do in postnatal life. The study of the role of hormones in prenatal development is a difficult one, but various experimental procedures are being developed which allow investigation to proceed, and have already contributed much to our knowledge of the subject. Foetal endocrine glands can be removed surgically, or inactivated by irradiation or by chemical procedures. Much information can be acquired too from Nature's own 'experiments', such as the anencephalic monsters which provide information on the role of the foetal hypothalamo/pituitary axis, by a study of the abnormalities which arise in its absence. In vitro cultivation of gland rudiments is another source of information about the interactions of the developing endocrine glands and their secretions. The Pituitary Gland. The effects of extreme deficiency or complete lack of pituitary hormones have been studied in experimental rats and rabbits decapitated in utero, and in human anencephalics by Bearn (1968). In the experimental animals, decapitation three days before Caesarean section at full term in the rats, or seven days before full term in the rabbits, did not interfere with the growth rate or with the bony development of the foetuses, even though at this stage in development the pituitary is normally differentiated and growth hormone can be detected in it. These experimental findings are in accord with the known fact that skeletal growth in human anencephalics is not retarded. In their case, the hypothalamus is definitely absent, but careful search often reveals the presence of the anterior hypophysis, usually small and underdeveloped. It has been said that these hypophyses contain substantial amounts of ACTH. As it is the function of the pituitary to form hormones, and the function of the hypothalamus to modulate their release, it is possible that the undoubted hypoplastic changes in target organs in anencephalics are due to failure in the hypothalamic release mechanism, or to extreme shortage of ACTH in the circulating blood. Experimental hypophysectomy causes a marked drop in foetal liver glycogen, possibly due indirectly to lack of glucocorticoid stimulus. The situation can be remedied by injections of ACTH. The foetal pituitary-thyroid axis has been the subject of much study. Several investigators have found that the embryonic thyroid gland is able to differentiate independently of the pituitary, but that in the absence of the pituitary stimulus, excretion of thyroxine is much reduced. The pituitary-adrenal axis is well developed in the prenatal period, although after birth in the rat there is a temporary state of refractoriness to adrenalin stimulation. Hypophysectomized embryos have very underdeveloped adrenal cortices. If however, these animals are given adrenocorticotropin the adrenals develop normally. Bearn (loc. cit.) found that the adrenal weights of rats decapitated four days before term were less than half those of their litter mates and much less well vascularised. An interesting finding in his study was that the thymus weights in decapitated foetuses were much increased. They could be kept down to normal weight by injections of ACTH. The doses of ACTH used in the Bearn's experimental rats were admittedly excessive and produced overgrowth of the adrenals; in these circumstances the thymuses were reduced in size in comparison with normal thymuses. The conclusions therefore were that the adrenals were under pituitary control, and also that the adrenals provide an inhibitory influence on thymus growth. Reduction in size of the foetal thymus also follows the injection of corticosteroid into the mother. The effects of hormones on the immunological apparatus of the body might be considered as by-products of endocrine research. The toxic effects of cortisone on lymphocytes, the inhibitory effects of stress on the production of antibody, and the stimulation of phagocytosis by oestrone and progesterone are fascinating by-ways which invite exploration. To return now to the adrenal, the foetal zone which normaily comprises four-fifths of the cortex, is almost absent in anencephalics. The fact that adrenal hypoplasia should occur in both man and experimental animals in these circumstances suggests that maternal ACTH is not able to cross the placental barrier. Studies on the hypophyseal-gonadal axis show that gonadotropic stimulation is not essential for the differentiation of the genital tracts, but at. a certain late stage in pregnancy (22-24 days in the rabbit) embryonic hypophyseal gonadotropin is essential for the normal functioning of the testis. At a later stage in development, the hypophysis again becomes inessential. Histologically the gonadotropin producing cells can be shown to be in a high state of activity in the few days preceding their requirement for testicular development. Thereafter, they become relatively inactive. The Thyroid Gland. The development of the foetal thyroid takes place between the eighth and fourteenth week in man. There is some evidence that the thyroid can abstract labelled iodine from the blood before follicles are visible histologically. The time of development of the thyroid in animals varies with the species, being earlier in those animals and birds born mature than in those born immature. In the sheep it is well differentiated at the 50th day out of 150 days total gestation, in the rat at the 18th day out of 21. Elimination of foetal thyroid function has a profound effect on the embryo, which is perhaps even more obvious in birds than in mammals. The yolk sac remains outside the body cavity, instead of being drawn within, as is usual. There is delay in the maturation of ossification centres and the growth of the embryonic plumage is much retarded. Likewise, in mammals there is delayed development. Lascelles and Setchell (1959) demonstrated the delay in bony development and maturity in lambs born to sheep treated with methylthiouracil, a drug which lowers thyroid function. The treatment seemed to reduce the protein bound iodine in the foetus much more than in the dam. As in other experimental animals, thyroid depletion caused an increase in the lipid content of the animals. Fat accumulated within the foetuses and the foetal plasma showed increases in all lipid fractions. Injection of thyroxin into affected animals restored the situation to normal by limiting fat accumulation. A more important result of foetal thyroid inactivity however, is the effect on the central nervous system. The congenital cretinism arising in the children of hypothyroid mothers can be duplicated experimentally in rabbits and rats. Changes in the cerebral cortex in the hypothyroid foetus include decreased size of neurones and defective myelination. If the subjects are not treated in the very early days of post-natal life, then the brain damage becomes irreversible. Endemic and enzootic cretinism is associated with skeletal malformations as well as with congenital goitre. Morphological changes have been noted in experimentally hypothyroid animals such as persistent foramen ovale in piglets. Rats develop the eye abnormalities which are associated with A deficiency, namely cataract, coloboma and occasionally anophthalmia. Cleft lip and cleft palate are common. Low succinic dehydrogenase activity, an indication of enzyme inhibition in hypothyroidism, can be restored to normal if the missing hormone is replaced early in life, but not later than three weeks after birth. The injection of excessive pituitary thyroid-stimulating. hormone into certain strains of rats causes hydronephrosis and hydroureters resembling that produced by giving excess vitamin A. The similarity between the eye lesions of hypothyroidism and of A deficiency recall the fact that several investigators have stated that in hypothyroidism the absorption of carotene and its conversion to vitamin A are decreased. The Panereatic Islets. Insulin and the associated blood glucose levels are among the most important requirements for maintaining a steady and normal rate of development in the foetus. In man, the pancreatic islets start to differentiate about the 7th week and by the 20th week both alpha and beta cells are present, in association with the capillaries necessary for circulating their secretion. Developmental abnormalities arising in diabetes have already been mentioned in connection with variations in maternal vitamin A status. The injection of insulin into experimental animals also causes foetal deformities in rabbits and in chicks. Following the injection of insulin on the fourth day of incubation, chick embryos become hypoglycaemic. When they hatch, they show various skeletal abnormalities including rumplessness and a syrenoid condition (fusion of the lower limbs). The Adrenal Gland. Both the adrenal cortex and the medulla produce their secretions in intrauterine life and are important for normal foetal development. In man the differentiated foetal cortices are already showing lipid accumulation by the tenth or twelfth week; the medullary chromaffin reaction becomes apparent at about 15 weeks. At midterm, the adrenal cortex is able to synthesize a variety of steroids, as is the foetal liver, which at this time has some claim to the status of an endocrine organ in this respect. The placenta too is able to convert pregnenolone to progesterone in the preparatory stage of steroid production (Solomon et al., 1967). The chick embryo adrenal gland becomes visible by the 11th or l2th day of incubation. Differentiation in both man and birds is not under pituitary control until a fairly late stage, when the pituitary stimulus accelerates the growth of the gland and stimulates the accumulation of steroid precursors in the zona fasciculata. In the absence of the pituitary further growth is retarded. The functional ability of the foetal adrenal is well demonstrated by the fact that removal of one adrenal stimulates overgrowth of the other. Just as in post-natal life, the compensatory hypertrophy is prevented if the animal is supplied with cortisone. Prolonged supply of exogenous cortisone retards adrenal development by inhibiting the production of ACTH and thus interfering with the negative feedback mechanism. A common test used to detect adrenal activity is the increase in duodenal alkaline phosphatase in embryos. This enzyme increases markedly in the mouse duodenum from the 15th intrauterine day. The increase can be prevented by foetal adrenalectomy at the l2th intrauterine day. This suggests that the adrenal cortical secretion is important in the differentiation of the duodenal epithelium. Adrenal'' control of duodenal phosphatase is also exerted post-natally and it may be that the adrenal exhaustion following stress of various types has a contributory effect in impairing the function of the mucosa in duodenal ulceration. In the rat, cortisone stimulates the synthesis of alkaline phosphatase both in the duodenal microvilli and in the brush borders of the renal tubules. Cleft palates can be induced by treating pregnant mice with cortisone or hydrocortisone from about the 11th to the l4th gestational day. The incidence varies with the strain of mouse used and may attain 100%. That the production of cleft palate by cortisone is not purely of experimental interest is demonstrated by the fact that 1% of the babies born to pregnant women treated with cortisone have cleft palates - a significant increase over the incidence in women not so treated. In animals the adrenal medulla is known to have the ability to secrete catecholamines before birth. Extra-adrenal chromaffin bodies secrete only noradrenalin. The intra-adrenal chromaffin tissue is able to convert noradrenalin to adrenalin before birth in both man and rats. The enzyme which catalyses the conversion, phenylethanolamine N-methyl transferase is under the control of the pituitary gland. (It will be remembered from Chapter 5, that vitamin B12 is a coenzyme for the reaction.) In rats deprived of the pituitary by decapitation on the l7th day of gestation, the activity of this enzyme drops by about 80 %. Not only ACTH, but also hydrocortisone acetate, has the ability to restore the activity towards normal. It seems therefore, that both the pituitary and the adrenal cortex have a modifying effect on the adrenalin/noradrenalin ratio of the medulla. The dramatic fall in enzymatic activity following foetal pituitary ablation suggests that maternal ACTH is not able to cross the placenta to substitute for the lost foetal hormone. If adrenalin is given in excess to rat or rabbit foetuses, it causes degenerative changes in the foetal vascular system. As a result, some tissues become necrotic, and there may even be prenatal loss of limbs or tails. The Gonads. Disturbances in the development of the gonads are known to occur as a result of the therapeutic use of sex hormones during pregnancy, or as a result of pathological processes occurring in the maternal gonads. In the male rat, the testicular interstitial cells are histologically demonstrable about the l5th day of gestation, in man about the l6th week. In the female the analogous periods for ovarian development are the first day of post-natal life in the rat and the 30th week in the female human foetus. Secretion by testicular and ovarian endocrine cells is stimulated by pituitary gonadotropin, and synthesis requires nicotinamide coenzymes, vitamin A and possibly other vitamins. Hypophysectomy leads to a reduction in the number of interstitial cells present in the testis and arrested development of the male genital tract. Genetic sex is determined at the moment of conception. Gonadal sex is not determined until a later period. Somatic sex depends on the development, or the absence of the testicular androgens in mammals. The sequence of development in the rabbit provides an illustration of the process. The embryonic gonads are recognisable by about the l5th day of gestation, and they are identical in both sexes. In the presence of the functioning pituitary, the gonad in a genetic male then begins to secrete androgen. This is followed by regression of the Mullerian duct and the development of prostatic rudiments. Full development of somatic masculinity occurs when the internal and external genital apparatus and the secondary sex characters appear. Loss of testicular activity just before gonadal sex differentiation, by castration or by chemical means, inhibits the process, and the animal develops along the female line of differentiation. In such a case, a genetic male acquires feminine characteristics and is therefore pseudo-hermaphrodite. The gonadectomised genetic female suffers no such disability, and continues to develop along the female pattern. We must conclude therefore, that foetal testicular hormones are essential for the development of the portion of the Wolffian duct which forms the vas deferens, and for the formation of prostatic anlagen from the urogenital sinus. The interstitial cells of the embryonic testis are thus the major factor in deciding somatic sex differentiation in mammals. In the rat, it is known that the ability of foetal testicular tissue to make testosterone from progesterone is at its height at about 18.5 days gestation. The vitamin-containing coenzymes necessary for steroid biosynthesis will therefore be required for several days prior to this time. Sexual development in birds differs from that in mammals in that the ovarian hormones have the controlling influence in most cases investigated. This is possibly related to the fact that in mammals the males are the heterozygous sex (possessing an X and a Y chromosome), and in birds the males are homozygous (possessing two X chromosomes). Human cases of bilateral ovarian agenesis (Turner's syndrome), at one time thought to involve chromosomal females only, are now known to include both genetic males and genetic females. The lack of testicular hormone in the genetic males apparently allows the formation of the infantile female type of genitalia and secondary sex characters, as in the experimental rabbit. Turner's syndrome in man is associated with anomalies of the central nervous system and the cardiovascular and skeletal systems. Chromosomal abnormalities Most of those agents which were mentioned in the previous chapter as causing chromosomal abnormalities and carcinogenesis are able, in the foetus, to produce teratogenesis; ionizing radiations, viruses and antimetabolites are all teratogenic. Genetically determined foetal abnormalities are by now well recognised. In some cases the chromosomal damage is visible to the electronically aided eye, in other cases the genetic defect can be traced only by an examination of family history. Some genetic abnormalities can be duplicated exactly by environmental changes in utero, and these are known as phenocopies, that is, they are non-hereditary defects mimicking the defects induced by mutant genes. Examples are the syndactyly, cleft palate and coloboma induced by A deficiency, riboflavin deficiency or folic acid deficiency. The purines and pyrimidines, which require folic acid for. their synthesis, form part of the prosthetic groups of enzymes, the absence of which may cause foetal abnormality. Mercaptopurine, for example, which is used in the treatment of human leukaemia, if given to pregnant rats, induces foetal abnormalities, including cleft lip and palate, deformed limbs, malformation of the urogenital and cardiovascular systems and of the adrenals (Kury et al., 1968). It is important to distinguish between phenocopies and true genetic defects. Ingalls, Ingenito and Curley (1964), while investigating the teratogenic effects of a nicotinamide antagonist in mice, were able to show that the malformed foetuses had a high proportion of chromosomal abnormalities as compared with normal foetuses. Polyploid cells and cells with fragmented chromosomes were found not only close to the palatal defect which occurred in 95 % of the test animals, but also at some distance from the lesions. Examination of the maternal bone marrow cells also revealed a rise in chromosomal abnormalities as compared with the normal control animals. Six days after the injection of the teratogen 56 % of the maternal bone marrow cells showed fragmentation of the chromosomes and 6 % were polyploid. Had the previous history of the animals not been known accurately, the chromosomal anomalies and congenital malformations of the foetuses would have been interpreted as true hereditary defects. Here we have proof that the genetic substance itself is susceptible to environmental injury. It remains to be seen how far-reaching the effects of such damage may be - whether they may be passed on to succeeding generations in a latent or an overt form. As in neoplasia, so in teratogenesis, karyotypes may not always be visibly abnormal, but the possibility remains that mutations not yet visible by present microscopic methods nevertheless do exist. Such alterations may provide the molecular lesion which causes eventually the production of tumours or malformations. Down's syndrome, or mongolism, provides the obvious link-up between teratogenesis and neoplasia. The affected persons have an incidence of leukaemia grossly in excess of that of other people. Genetic defects are not always transmitted by the maternal chromosomes. The paternal contribution, although contained in one cell only, nevertheless provides half the programme for the fashioning of the new organism, and must be held responsible for the defects as well as the good qualities of the progeny. This can be illustrated experimentally by treating rabbit sperm with colchicine. The foetuses of rabbits impregnated with this sperm show various anomalies (Chang, 1944). Similarly a paternal chromosomal defect has been suspected as being the cause of multiple anomalies in a human infant. In a case reported by Day et al (1967), the mother had a normal karyotype, but the father and child had part of the 18th chromosome missing. Physically, the father was apparently normal; the infant had various abnormalities including microcephaly, cryptorchidism, hypospadias and talipes. Damage to paternal chromosomes as a cause of foetal anomaly is a fairly new concept in human medicine and one which has been slow to gain acceptance. In the field of animal medicine, economic considerations have ensured that the nutritional requirement of both the sire and the dam are held of equal importance for the development of healthy progeny. Two articles of general interest, not mentioned in this chapter are:-
REFERENCES:- Adamstone F.B. (1931), The effect of Vitamin E deficiency on the development of the chick. Journal of Morphology, 52, pp. 47-89. Bearn J.G. (1968), The thymus and the pituitary-adrenal axis in anencephaly. British Journal of Experimental Pathology, 49, pp. 136-144. Carter C.O. (1968), The genetics of congenital malformations. Proceedings of the Royal Society of Medicine, 61, pp. 991-995 Chang M.C. (1944), Artificial production of monstrosities in the rabbit. Nature 154, p.150 Davison A.N. , Dobbing J. (1966), Myelination as a vulnerable period in brain development. British Medical Journal, 22, pp.40-44. Day E.J., Marshall R., McDonald P.A.C., Davidson W.M. (1967) Deleted chromosome 18 with paternal mosaicism. Lancet, 2, p.1307 Ingalls T.H., Ingenito E.F., Curley F.J. (1964), Acquired chromosomal anomalies induced in mice by a known teratogen. Journal of the American Medical Association, 187 pp. 836-838. Kury G., Chaube S., Murphy M.L. (1968)Teratogenic effects of some purine analogues in fetal rats. Archives of Pathology, 86, pp. 395-402 Lascelles A.K., Setchell B.P.(1959) Hypothyroidism in the sheep. Australian Journal of Biological Sciences, 12, pp. 455-465 Nelson M.M., Evans H.M. (1956), Failure of ovarian hormones to maintain pregnancy in rats deficient in pantothenic or pteroylglutamic acid. Proceedings of the Society of Experimental Biology and Medicine, 91, pp. 614-617 Nelson M.M., Wright H.V., Asling C.W., Evans H.M. (1955), Multiple congenital abnormalities resulting from transitory deficiency of pteroylglutamic acid during gestation in rats Journal of Nutrition 56, pp. 349-363 Otis E.M., Brent R. (1954), Equivalent ages in mouse and human embryos. Anatomical Record, 120, pp.33-63 Pinsky L., Fraser F.C.(1960), Congenital Malformations after a two hour inactivation of nicotinamide in pregnant mice. British Medical Journal, 2, pp.195-197 Solomon S., Bird C.E., Ling W., Iwamiya M., Young P.C.M.(1962), Formation and metabolism of steroid in the foetus and placenta.Recent Progress in Hormone Research, 23, pp. 297-335 Tonutti E., Fetzer S.(1956), Über entwicklung und differenzierung der glandotrop gesteurten inkretorishen Gewebe beim Menschen. 3. Symposium. Deutsche Geselschaft für Endocrinologie. Springer, Berlin, pp.1 - 12
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