BritishJournal of Haematology, 1972, 22, 287.

A Study of Storage Iron in the Placental and Foetal Tissues of the Rat

NATHAN KAUFMAN AND JOHN C. W n m

Department ofpathology, Queen’s University, and Kingston General Hospital, Kingston, Ontario, Canada

(Received 12 M a y 1971 ; acceptedfor publication 28 June 1971)

SUMMARY. Part of the iron entering the placenta late in gestation is stored as ferri- tin and haemosiderin; the function of placental storage iron is not known. 50-60% of foetal iron is found within storage compounds. There are relatively few studies of the foetal tissues responsible for the synthesis of storage iron-protein.

In order to investigate these aspects of iron metabolism we measured the quantity and concentration of ferritin and haemosiderin iron in the placental and foetal tissues of the rat during the latter part of gestation; the incorporation of 59Fe into these compounds was also investigated.

Placental ferritin and haemosiderin iron reached peak values on day 17. A 40% decrease in the quantity of ferritin iron by day 19, without a corresponding change in placental weight, indicated mobilization of iron from placental ferritin; haemo- siderin iron remained unchanged. On day 21 (parturition) both iron storage com- pounds again increased in quantity. The amounts of storage iron deposited in the allantoic placenta and yolk sac were similar until day 21 when the former tissue con- tained 75% of the total quantity. The concentration of ferritin iron was higher in the yolk sac on days 17 and 19.

The major portion of foetal ferritin and haemosiderin was deposited late in ges- tation. The carcass and liver were the principal sites involved although small amounts were found in the spleen and gastrointestinal tract. The incorporation of 59Fe by ferritin and haemosiderin confirmed the results obtained by chemical analysis and also showed that there was a steady addition of iron to both compounds through- out the latter part of gestation.

Practically all the iron required by the foetus is transferred from the maternal blood across the placenta in the latter part of gestation (Bothwell & Finch, 1962) ; in the rat, for instance, the maximum rate of placental iron transport is observed between days 15 and 21 (Nylander, 1953; Glasser et a/ , 1968; Kaufman & Wyllie, 1970). Although most of the iron entering the placenta is deposited in foetal tissues, some accumulates in storage compounds (ferritin and haemosiderin) within the placenta. The function of this storage iron is not clear. Ferritin may regulate the movement of iron across the placenta (Nylander, 1953). Iron may also be stored temporarily in the placenta, when adequate amounts are available from maternal sources, in order to supplement the foetal supply at a later date. The iron which reaches the

Correspondence : Dr John C. Wyllie, Department of Pathology, Queen’s University, Kingston, Ontario, Canada.

C 287

288 Nathan Kaufman andJohn C. Wyllie

foetus is used principally for haemoglobin and storage iron-protein synthesis (Bothwell et al, 1958; Morgan, 1961); iron is known to accumulate in the liver and spleen during foetal development (Lintzei et al, 1 9 4 (quoted by Bothwell & Finch, 1962) ; Widdowson & Spray,

The objectives of this study were to investigate the formation and fate of storage iron- protein in the placenta and to determine the distribution of storage iron in the foetus of the rat. Ferritin and haemosiderin iron in the placental and foetal tissues were measured at various intervals during the later half of gestation; in addition, the incorporation of s9Fe into these compounds was examined.

1951).

MATERIALS AND METHODS

Pregnant Wistar rats, weighing 200-250 g, were fed laboratory chow and water ad libitum and maintained under standard animal care conditions. The timing of gestational age was based on the observation of spermatozoa in vaginal smears. The animals were used in groups of 16 at 12, IS, 17, 19, 20 and 21 days of gestation. The rats were anaesthetized with ether and injected, by way of the lateral marginal leg vein, with 20 pCi of s9Fe (s9Ferric chloride, concentration 20 pCi/ml and specific activity 8-12 mCi/mg Fe, Charles E. Frosst & Co., Montreal, Canada). 2 or 24 hr later the rats were dissected under ether anaesthesia to obtain the placentae and foetuses. From days 17 to 21 placental tissue was separated into allantoic placentae and yolk sacs; in addition, five foetuses from each litter were dissected to obtain liver, spleen, gastrointestinal tract and carcass (all remaining tissue) and the remaining foetuses kept intact. The foetal spleen was not included on day 17 because its minute size made the measurement of storage iron difficult. The tissues were weighed, chilled and homogenized with distilled water in a Potter-Elvejhem homogenizer. Ferritin and haemosiderin iron were measured by a modification of Kaldor's (1958) technique; 2,4,6-Tri (2'-pyridyl)-S-triazine was used in place of the orthophenanthroline reagent. The results were expressed as follows: (a) total ferritin and haemosiderin iron in mg; and (b) concentration of ferritin and haemo- siderin iron, in pg/g of tissue, in the placentae and foetuses obtained from each litter (and, where applicable, their respective subdivisions) at the various stages of gestation selected. The percentage of storage iron in the form of ferritin was calculated. The radioactivity (cpm) of an aliquot of ferritin and haemosiderin iron, extracted from placental and foetal tissue, was counted in a well-type solid scintillation counter (Nuclear-Chicago Corporation, Des Plaines, Illinois, U.S.A.) ; the total radioactivity in the form of ferritin and haemosiderin iron was determined for the placental and foetal tissue obtained from each litter at the various gestational stages. Mean values were calculated; where required, significance was assessed by the t-test according to the method of Goldstein (1964).

RESULTS Placenta

The mean weight of the placenta at term (day 21) was 7.27 g; there was a 77% increase in placental weight between days 15 and 21 (Fig I). 0.5 mg of ferritin iron was present in the placenta on day 17-a tenfold increase above the day 12 value (Fig I). 85% of this amount

289 Storage Iron in Placental and Foetal Tissues

was deposited between days 15 and 17; there was a 3 8% increase in the weight of the placenta during this interval (Fig I). By day 19, however, the level of ferritin iron had fallen to 0.3

,18

I I

12

Placenta*

d

Foefus*

T

17 19 20 ZI

Y7

Day of gestation

FIG I. Total quantity of femtin iron (open columns) and haemosiderin iron (hatched columns), in mg, in the rat placenta; placental weight (0). Each point represents eight pregnant animals. Vertical bars represent one standard error of the mean above and below the mean values.

TAE~LB I. Ferritin iron expressed as a percentage of total storage iron

Day of gestation

I2

1s I7 I9 20 21

82.9 f 2.3 (8) 84.7-+ 1.9 (8) 85.5f2.2 (8) 78.3 k 3.2 (8) 78.052.4 (8) 78.022.2 (8)

77.5 k 2.9 (8) 80.8f2.1 (8) 81.9k2.4 (8) 77-5 f 2.1 (8) 82.0f 3.2 (8) 78.7f2.5 (8)

* Mean f I SE of the mean; number of preg- nant rats used are given in parentheses.

mg-a decrease of 40% (P<o.oI); no further change was observed on day 20 (Fig I). The percentage of storage iron in the form of ferritin decreased from 85.5 on day 17 to 78.3 on day 19 (0.05 > P> 0.01) (Table I). The concentration of ferritin iron in the placenta was highest

290 Nathan Kaufman andJohn C. Wyllie

0.0~2+0.008 (8) 0.069+0.007 (8) 0.227+0.022 (8) 0.116+0.017 (8) 0.072+0.006 (8) 0.071 f0.006 (8)

on day 17; lower values were observed on days 1g,20 and 21 (Table 11). There was no change in mean placental weight between days 17 and 20; on day 21 an increase in weight and in the quantity of ferritin (0.6 mg) was observed (Fig I). Haemosiderin iron was also deposited in

TABLE II. Concentration of femtin and haemosiderin iron in the total placental and foetal tissues of the rat

0.012+0.002 (8) 0.011 +O.OOI (8) o.o+++o.oog (8) 0.035+0.006 (8) 0.020+0.003 (8) 0.017+0.00z (8)

Day 4 gestation

I2

I S I7 I9 20 21

Placenta*

0.042+0.008 (8) 0.064 + 0.008 (8) 0.117+0.010 (8) 0.083 + 0.005 (8) 0.069 f 0.008 (8) 0.060fo.009 (8)

0.011 + 0.003 (8) 0.015 + 0.001 (8) 0.021 + 0.002 (8) 0.019 + 0.004 (8) 0.019+0.004 (8) 0.017+0.004 (8)

I

* Mean + I SE of the mean; number of pregnant rats used is given in parentheses.

160

150 - 140 - 130 -

I x )

110-

-

-

g loo-

: 00- 4 c 90- .Q

- c

0 5 70-

- 60-

50-

40 -

c

30 -

I I I I 17 19 20 21

Doy of gestation

FIG 2. Concentration of ferritin iron in the allantoic placenta (A) and yolk sac (A), and of haemo- siderin iron in the allantoic placenta (0) and yolk sac (0) in pg/g of tissue. Each point represents eight pregnant rats. Vertical bars represent one standard error of the mean above and below the mean values.

Storage Iron in Placental and Foetal Tissues 291

the placenta during the latter half of gestation although in lesser amounts (Fig I). The quantity of haemosiderin iron increased to 0.08 mg on day 17 but, in contrast to ferritin iron, did not fall on day 19 (Fig I). A further rise was observed on day 21 (Fig I). The concentration of haemosiderin iron was highest on day :17 and decreased on day 20 (0.05 > P> 0.01) (Table 11).

The quantities of ferritin and haemosiderin iron present in the allantoic placenta and yolk sac between days 17 and 21 followed the pattern observed in the intact placenta. Thus ferritin

Day of gestation

FIG 3. Incorporation of s9Fe (cpmx IO-") into placental ferritin iron, 2 hr (A) and 24 hr (A) post- injection, and into placental haemosiderin iron, 2 hr (0) and 24 hr (.) post-injection. Each point represents eight pregnant rats. Vertical bars represent one standard error of the mean above and below the mean values.

iron was reduced on day 19 whereas haemosiderin iron remained unaltered. Similar amounts were present in each placental subdivision on days 17, 19 and 20; on day 21, however, the allantoic placenta contained about 75% of the placental storage iron due to an increased deposition of both ferritin and haemosiderin iron. The concentration of ferritin iron was highest in each placental subdivision on clay 17 but dropped on day 19 and thereafter remained at a low level (Fig 2). The yolk sac contained a higher concentration of ferritin iron than the allantoic placenta on days 17 and rg (Fig 2). The concentration of haemosiderin iron was low in both the allantoic placenta and the yolk sac; slightly higher values were observed in the yolk sac on days 19 and 20 (Fig 2). Neither placental subdivision increased in weight between days 17 and 19; on day 21 the allantoic placenta accounted for two-thirds of the placental mass.

292 Nathan Kaufman andJohn C. Wyllie

A small quantity of 59Fe was incorporated into placental ferritin iron on day 12; there was no difference between the values obtained 2 and 24 hr post-injection (Fig 3). "Fe incorpora- tion rose between days 12 and 15 (Fig 3). On day 17 the quantity of ferritin 59Fe, 24 hr post- injection, was about 56% lower (0.05 > P> 0.01) than the 2 hr value (Fig 3). On days 19 and 20 the 2 and 24 hr values were again similar; both showed a downward trend on the latter date (Fig 3). The incorporation of 59Fe into placental haemosiderin iron was very low on day 12; an increase in uptake was observed up to day 19, 2 hr after injection, and up to day 17,

3.0

3 1

Day of gestation

FIG 4. Total quantity of ferritin iron (open columns) and haemosiderin iron (hatched columns), in mg, in foetal rat tissues; total foetal weight (a). Each point, except for day 21, represents eight pregnant animals; day 21 values were obtained from seven pregnant animals. Vertical bars represent one standard error of the mean above and below the mean values.

24 hr after injection (Fig 3). There was no significant difference between the 2 and 24 hr values from days 12 to 20 (Fig 3). The incorporation of "Fe into ferritin and haemosiderin iron of the allantoic placenta and yolk sac, on days 17, 19 and 20, paralleled the quantities of storage iron determined chemically.

Foetuses The mean weight of the entire foetal mass at term was 41.74 g; 80% of the weight increase

between days 12 and 21 occurred in the last 5 days of gestation (Fig 4). Small amounts of ferritin and haemosiderin iron were present in the foetal tissues on days 12 (Fig 4). By day 19 about 50% of the final quantity of ferritin iron had been deposited; the remaining 50% was deposited in the last 24 hr of gestation (Fig 4). The deposition of haemosiderin iron followed

293 Storage Iron in Placental and Foetal Tissues

a similar pattern (Fig 4). Ferritin iron composed about 80% of the total storage iron; no significant change in the ratio of ferritin iron to total storage iron was observed from days 12

to 21 (Table I). The concentration of ferritin iron in the foetal tissues was highest on day 17 (Table 11). The concentration of haemosiderin iron did not change between days 12 and 21

(Table 11).

1.2 -

1.1 -

1.0 -

0.9 -

0 8 -

- 0.7- z u

.- 0.6-

.= 8 0.5- .:

E +

T T

0.5,-

T

GI Spleen

0 Gastrointestlnal tract

Liver

B3 Carcass

T T

Day of gestation

FIG 5 . Total quantity of ferritin and haemosiderin iron, in mg, in the foetal rat spleen, gastrointestinal tract, liver and carcass (the spleen was not included in the day 17 analysis). Each point represents eight pregnant animals. Vertical bars represent one standard error of the mean above and below the mean values.

Ferritin and haemosiderin iron were measured in the foetal liver, gastrointestinal tract and carcass on days 17, 19,20 and 21 and in the foetal spleen on the latter three days. Ferritin iron exceeded haemosiderin iron in quantity at each interval (Fig 5) . The carcass was the principa1 site of accumulation for both ferritin and haemosiderin iron; quantities present on day 21 were about 50% greater than the day 17 values (Fig 5). Lesser amounts of ferritin and haemosiderin iron were present in the liver (about 55% and 25% respectively of the carcass content); there was an increase in ferritin iron between day 17 and 21 (0.05 > P> 0.01) but not in haemosiderin iron (Fig 5) . The quantities of ferritin and haemosiderin iron in the gastrointestinal tract and

294 Nathan Kaufman andlohn C. Wyh'e

spleen were small and did not increase between days 17 to 21 and days 19 to 21 respectively

59Fe was incorporated into ferritin and haemosiderin iron of the total foetal mass, both at 2 and 24 hr (values were similar), from day 12 to 21. The pattern of "Fe uptake corre- sponded to the distribution of storage iron measured chemically (Fig 4). There was little

Pig 5).

Day of gestation

FIG 6. Incorporation of "Fe (cpmx I O - ~ ) into ferritin and haemosiderin iron of rat foetal spleen, gastrointestinal tract, liver and carcass (the spleen was not included in the day 17 analysis). Each point represents six pregnant animals. Vertical bars represent one standard error of the mean above and below the mean values.

uptake of "Fe by either ferritin or haemosiderin iron on day 12; increased amounts were incorporated into both compounds between days 12 and IS, 15 and 17 and, in the case of ferritin, between days 17 and 21 (o.o~> P> 0.01).

"Fe uptake by ferritin and haemosiderin iron in the foetal liver, gastrointestinal tract and carcass from days 17 to 21 and in the foetal spleen from days 19 to 21,24 hr subsequent to injection, is presented in Fig 6 (the 2 hr values were similar). The carcass and liver were the principal sites of deposition of storage iron; there was little difference in the amount of ferritin 'Fe accumulated although more haemosiderin 59 Fe was present in the carcass than

295 Storage Iron in Placental and Foetal Tissues

in the liver (Pc 0.01) (Fig 6). The incorporation of 59Fe into hepatic ferritin and haemosiderin iron rose between days 17 and 19 (Fig 6). The spleen and gastrointestinal tract contained small quantities of storage 59Fe (Fig 6).

DISCUSSION

Large quantities of iron cross the rat placenta to the foetal tissues during the final 6 days of gestation (Glasser et al, 1968; Kaufman & Wyllie, 1g7o)-an interval marked by accelerated foetal and placental development. Our results show that part of the iron entering the placenta is retained in storage compounds. The amount ofstorage iron is not insignificant; for example, on days 17 and 21, the placenta contained 36% and 19% respectively of the ferritin iron present in the foetal and placental tissues. There was a peak in ferritin and haemosiderin iron deposition in the placenta on day 17; by day 19, however, a 40% reduction in ferritin iron had occurred. The percentage of storage iron in the form of ferritin and the concentration of ferritin iron also decreased; haemosiderin iron remained unaltered. There was no change in placental weight during this period. On day 21 (immediately prior to parturition) we ob- served a further increase in placental ferritin and haemosiderin iron which restored the former compound to the day 17 level. Placental weight also increased during the final 24 hr of gestation. Storage iron was divided about equally between the allantoic placenta and the yolk sac from days 17 to 21 ; the concentration of ferritin iron was higher in the yolk sac on days 17 and 19.

Our results show that part of a tracer dose of 59Fe injected into the maternal circulation is incorporated into placental ferritin and haemosiderin iron. We observed that the quantity of 59Fe present in placental ferritin at 24 hr on day 17 was about 56% lower than the 2 hr value. The 2 and 24 hr levels of placental ferritin 59Fe did not differ significantly thereafter; a downward trend in both values was observed on day 20. Small quantities of 59Fe were incorporated into placental haemosiderin over the same period; the 2 and 24 hr values, on each date of analysis, did not differ significantly. The pattern of storage 59Fe uptake in the allantoic placenta and the yolk sac confirmed the findings obtained by chemical analysis.

Iron is known to accumulate in the placenta during pregnancy; Wislocki et a2 (1946) and Nylander (1953) demonstrated iron deposits in both allantoic placenta and yolk sac using histological techniques. The retention of small amounts of 59Fe in the rat placenta was observed by Glasser et a2 (1968) and in the human placenta by Fletcher & Suter (1969). Nylander (1953) and Morgan (1961) have measured placental storage iron in the rat at various times in late gestation. Their findings differ from ours in that storage iron was observed to rise to a maximum level about days 17-19 and thereafter remain unchanged; the concen- tration of storage iron fell on days 20 and 21 as a result of the increasing placental mass. Nylander (1953) suggested that iron may cycle through placental ferritin prior to reaching the foetus; thus the transfer of iron across the placenta could be regulated by ferritin. The rapidity of placental iron transfer (Bothwell et al, 1958; Davies et al, 1959; Glasser et al, 1968; Fletcher & Suter, 1969; Kaufinan & Wyllie, 1g70), however, would argue against such a process. It appears to us that the placenta may pick up iron in excess of the immediate foetal demand at certain stages of pregnancy. It is not clear whether the capacity of the foetal plasma and tissue ‘receptors’ for iron is exceeded or whether the utilization of iron by the foetus lags

296 Nathan Kaufnian andJohn C. Wyllie

behind availability. It has been shown that the rat placenta continues to accumulate iron after the removal of the foetus (Glasser et al, 1968; Lane, 1968). Glasser et al(1968) state that iron uptake is an important primary function of the placenta. Iron, not transferred to the foetus, is therefore stored as ferritin and haemosiderin. Such a mechanism would prevent cellular damage from the accumulation of a large quantity of free iron which may poison intracellular enzymes (MacDonald, 1970). Our results indicate that part of the placental ferritin iron, from both the allantoic placenta and the yolk sac, is subsequently withdrawn between days 17 and 19 presumably for use by the foetus; haemosiderin iron does not appear available for this purpose. The reduction of the 24 hr level of ferritin 59Fe below the 2 hr value on day 17 also points to the mobilization of 59 Fe already incorporated into ferritin.

The yolk sac of the rat placenta, although separated by Reichert’s membrane from maternal plasma, is engaged with the transfer of iron to the foetus (Nylander, 1953 ; Glasser et al, 1968 ; Kaufman & Wyllie, 1970). Nylander’s (1953) measurements of the storage iron content of the allantoic placenta and yolk sac also demonstrated a higher concentration of storage iron in the latter; the epithelial cells of the visceral layer of the yolk sac stained more intensely for iron than did cells of the allantoic placenta (Nylander, 1953). These cells resemble absorptive epithelial cells lining the small intestine (Lambson, 1966; Carpenter & Fern, 1969). They may possess a greater capacity for ferritin synthesis than the cytotrophoblast of the allantoic placenta and thus acquire a higher concentration of ferritin iron. On day 21 we noted an increase in placental ferritin and haemosiderin; 75% of the storage iron was located in the allantoic placenta which comprised two-thirds of the placental mass on this date. It is of inter- est that Wislocki et a1 (1946) observed large amounts of nuclear iron and lesser amounts of cytoplasmic iron in cytotrophoblastic giant cells of the rat placenta at term.

The bulk of iron transferred to the foetus in the latter half of gestation is used either for haemoglobin synthesis or is stored as ferritin and haemosiderin; despite the magnitude of foetal erythropoiesis 50-60% of the foetal iron is in a storage form (Bothwell et al, 1958; Morgan, 1961). Our observations show that storage iron deposition takes place principally between days 17 and x t h e period of rapid foetal growth. Ferritin is the chief storage com- pound; the ratio of ferritin iron to haemosiderin iron is constant throughout the latter half of gestation. The concentration of ferritin iron in the foetus reached a maximum level on day 17; no further change was observed because increases in storage iron were balanced by the en- larging foetal mass. The carcass and liver were the principal sites of storage iron accumulation; smaller amounts were present in the gastrointestinal tract and spleen. Storage iron isolated from the carcass was probably derived from both the bone marrow and skeletal muscle; in the mature animal, storage iron is found in about equal amounts in the liver (one third), bone marrow and spleen (one third) and skeletal muscle (one third) (Bothwell & Finch, 1962). The relatively large proportion of foetal iron deposited in storage compounds is due to the iron requirements of neonatal life. Mammalian milk, in general, is low in iron (McCance & Widdowson, 1951).

59Fe, injected into the maternal circulation, crosses the placenta to the foetuses in increasing amounts as gestation advances (Glasser et al, 1968; Kaufman & Wyllie, 1970) ; a part is rapidly incorporated into foetal ferritin and haemosiderin for our observations show that the 2 hr uptake values do not differ significantly from the 24 hr levels. Our results show further that storage iron-protein is synthesized by the foetus throughout the latter part of gestation,

Storage Iron in Placental and Foetal Tissues 297 particularly as increasing amounts of iron are made available from the mother; although ferritin is the compound of preference there is a constant input into haemosiderin. There was a consistent pattern of storage 59Fe deposition in the foetal liver, spleen, gastrointestinal tract and carcass in late gestation as anticipated from the results of the chemical analysis; storage "Fe was found principally in the carcass and liver. In the human foetus, 59Fe accumulates first in the liver as storage iron and is probably mobilized at a latter date for hemoglobin synthesis (Fletcher & Suter, 1969).

ACKNOWLEDGMENT

This work was supported by the Medical Research Council, Ottawa, Canada.

REFERENCES

BOTHWELL, T.H. &FINCH, C.A. (1962) Iron Metabolism. Little, Brown, Boston.

BOTHWELL, T.H., PRIBILLA, W.F., MEBUST, W. & FINCH, C.A. (1958) Iron metabolism in the pregnant rabbit. Iron transport across the placenta. American Journal of Physiology, 193, 615.

CARPENTER, S.J. & FERM, V.H. (1969) Uptake and storage of thorotrast by the rodent yolk sac placenta: an electron microscopic study. American Journal of Anatomy, 125, 429.

DAVIES, J., BROWN, E.B., JR, STEWART, D., TERRY, C.W. & SISSON, J. (1959) Transfer of radioactive iron via the placenta and accessory fetal membranes in the rabbit. American Journal OfPhysiology, 197, 87.

FLETCHER, J. & SUTER, P.E.N. (1969) The transport of iron by the human placenta. Clinical Science, 36,209.

GLASSER, S.R., WRIGHT, C. & HEYSSEL, R.M. (1968) Transfer of iron across the placenta and fetal membranes in the rat. AmericanJournal OfPhysiology, 215, 205.

GOLDSTEIN, A. (1964) Biostatistics, p 51. Macmillan, New York.

KALDOR, I. (1958) Studies on intermediary iron meta- bolism. XII. Measurement of the iron derived from water soluble and water insoluble non-haem compounds (ferritin and haemosiderin iron) in liver and spleen. Australian Journal of Experimental Biology, 36, 173.

KAUFMAN, N. & WYLLIE, J.C. (1970) Matemofoetal iron transfer in the rat. BritishJournal ofHaematology,

LAMBSON, R.O. (1966) An electron microscopic visualization of transport across rat visceral yolk sac. American Journal of Anatomy, 118, 21.

LANE, R.S. (1968) Regulating factors in the transfer of iron across the rat placenta. British Journal of Haeniatology, 15, 365.

MACDONALD, R.A. (1970) Hemochromatosis: a perlustration. American Journal of Clinical Nutrition, 23, 592.

MCCANCE, R.A. & WIDDOWSON, E.M. (1951) The metabolism of iron during suckling. Journal of' physiology, 112.450.

MORGAN, E.H. (1961) Transfer of iron from the pregnant and lactating rat to the foetus and yomg. Journal OfPhysiology, 158, 573.

NYLANDER, G. (1953) On the placental transfer ofiron. An experimental study in the rat. Acta Physiologica Scandinavica, 29, Suppl. 107.

WIDDOWSON, E.M. & SPRAY, C.M. (1951) Chemical development in utero. Archives of Disease in Child- hood, 26, 205.

WISLOCKI, G.B., DEANE, H.W. & DEMPSEY, E.W. (1946) The histochemistry of the rodent's placenta. American Journal ofrlnafomy, 78, 281.

19, 51.5.