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Home , Epidermal growth factor, Insulin

Development 104, 137-145 (1988) 137 Printed in Great Britain © The Company of Biologists Limited 1988

The effect of epidermal , and transferrin on the growth-promoting properties of serum depleted by repeated culture of postimplantation rat embryos

M. K. PRATTEN, A. M. BROOKE, S. C. BROOME and F. BECK

Department of Anatomy, University of Leicester, Leicester LEI 7RH, UK

Summary

Homologous serum, when repeatedly used for the moters are originally present at very low concen- culture of postimplantation rat embryos, rapidly loses trations and become rate limiting when serum is its capacity to support growth and development. recycled. Many growth factors and fall into Replenishment of the 'exhausted' serum with this category and it is likely that a considerable and vitamins (MEM vitamin concentrate - Flow number are involved when serum is 'exhausted' by Laboratories) together with gentle dialysis to remove repeated use. When insulin, small molecular weight toxic metabolites (lactate etc) or rat transferrin are added to dialysed 'exhausted' fails to restore the growth-promoting properties of the serum each effects a partial restoration of growth of serum. This suggests that 'recycled' serum has been rat embryos. depleted of specific growth-promoting factors. Such serum that has been subjected to dialysis can be completely replenished by addition of 30 % normal rat Key words: rat embryo culture, recycled serum, growth serum. It is therefore probable that the growth pro- of embryos, growth factors.

Introduction Beck, 1984, 1985). Indeed, the fact that many such factors are of importance to embryonic growth is Although it has been apparent for some time that demonstrated by the evidence that fluid contained homologous serum has a finite growth-supporting within the cultured visceral yolk sac has some growth- capacity for postimplantation rat embryos cultured in promoting properties for rat embryos (Dunton et al. vitro, the important biochemical factors involved 1986). have not been effectively identified. Some dilution of It has been suggested by Klein et al. (1978) that the serum is possible whilst still maintaining normal certain specific are depleted from serum growth (Cockroft & Coppola, 1977; Huxham & Beck, when rat embryos are cultured at high density. Also 1985) but the studies performed indicate that 50% Sanyal (1980) has shown that rat embryos mediate serum is required for growth to be comparable to that changes in the components of the culture medium, observed in utero. When the rat serum is replaced by particularly in terms of pH, Pco> glucose, urea and serum, even with the addition of excess creatinine. More recently Priscott (Priscott et al. glucose, growth of rat embryos is not completely 1983) has to some degree extended the studies on normal unless a small proportion (10 %) of rat serum serum proteins and has tentatively identified the is added (Gupta & Beck, 1983; Lear et al. 1983). This proteins that are depleted by rat embryos in culture as implies that there are -specific factors in rat a^-macroglobulin and transferrin, amongst others. serum which the embryo requires. In this study, we have attempted to 'exhaust' the The nutritional role of the rat visceral yolk sac is growth-supporting capacity of rat serum by using it well established (Freeman et al. 1981). In addition, it for repeated culture. We have assessed the extent of is now clear that the yolk sac transports many embryonic growth and development with recycled macromolecules to the embryo intact (Huxham & serum and have attempted to elucidate whether the 138 M. K. Pratten and others limited growth observed was due to depletion of plemented with glucose and vitamins was also used with the 1 specific factors or to toxic changes in the serum addition of insulin (0-25 ng ml" ), epidermal growth factor brought about by the repeated culture. In addition, (0-25 ngml"') or rat transferrin. The extent of growth and the serum has been supplemented with factors that differentiation of embryos was assessed after a total of 50 h in culture as above. Epidermal growth factor (from mouse may be depleted during repeated culture in an at- submaxillary ) and insulin (porcine, crystalline, 0-5 % tempt to identify some of the specific agents involved. Zn) were obtained from Sigma Chemical Co. Ltd, Poole, Dorset, UK. Rat transferrin was prepared by the method of Schreiber et al. (1979). Materials and methods Biochemical methods Culture of rat embryos in whole and recycled rat The pH of samples of sera were measured with a Pye serum Unicam digital pH meter. Care was taken to keep samples Wistar rats were mated overnight and pregnancy was timed in airtight containers before measurements were performed from midnight preceding the morning on which vaginal to avoid any changes in pH. The osmolality was measured plugs were observed. Conceptuses were explanted at 9-5 using a Halbmikro osmometer. days (early head-fold stage, containing three somites), Lactate levels were measured using a diagnostic from according to the standard method of rodent embryo culture Sigma Chemical Co., glucose levels were measured by the described by New (1978). Briefly, explanted embryos were glucose oxidase method (kit from Sigma Chemical Co.) and cultured in homologous sera (see below) at 37°C in glass total was assessed by a modification of the Lowry bottles in a roller incubator. At the start of culture the method (Lowry et al. 1951). incubation bottles were gassed with 5 % O2: 5 % CO2: 90 % The levels of insulin in fresh and recycled sera were N2, after 24 h with 20% O2: 5 % CO2: 75 % N2 and after assayed using a double antibody radiolabelling method 44 h with 40 % O2: 5 % CO2: 55 % N2. (Wellcome Laboratories). Serum was prepared from both male and female rats by immediate centrifugation and stored at —18°C until used. Before culture the serum was heat-inactivated at 56°C for Results 30min. From a pool of approximately 50ml serum, 10ml was reserved as control serum and was treated in exactly the Effect of culture in recycled serum on embryonic same way as experimental serum except that it was cultured growth without embryos. In this way, the effects of incubation, freezing and thawing and filtration were eliminated. The Table 1 shows that the growth of embryos in serum remainder was used as culture medium for embryos from that has been recycled once is greatly retarded. If the 9-5-11-5 days of development at a density of 1 serum was used again hardly any embryonic growth embryo ml"'. After culture for 50 h the serum was frozen was obtained. Even when glucose (2mgml~') and and stored. It was then sterilized by passage through a vitamins (10jdml"' minimum essential medium con- R Millex-GV 0-22 urn filter unit (Millipore ) and reused for centrate) were added back to recycled serum, embry- culture of further embryos with or without the addition of 1 onic growth was severely impaired. Control embryos 2mgml~' glucose and 10^1 ml" MEM vitamin concentrate grown in serum subjected to the same regime of (Flow Laboratories). A small sample of medium was freezing and thawing grew normally. removed at each stage for biochemical analysis. Serum was recycled in this way until the culture of explants resulted in When recycled serum was dialysed (to remove any no significant growth as assessed by the morphological low molecular weight toxic waste products) and scoring system of Brown & Fabro (1981) which assesses the glucose and vitamins added, only limited improve- extent of growth and differentiation of 13 different embryo- ment in growth was observed, indicating that the loss logical features including development of neural tube, limb of growth-supporting properties can only be partially bud, , otic and optic systems and yolk sac. In addition, reversed in this way. After similar dialysis of whole measurements were made of protein levels (Lowry et al. serum embryonic growth was hardly affected in terms 1951), yolk sac diameter, crown-rump length and somite of morphological score or yolk sac diameter, although number. Such serum was considered to be 'exhausted'. there was some effect on the protein level of the 'Exhausted' serum and whole serum were then dialysed embryos. using Visking size 2 tubing (Medicell International Ltd, When recycled serum is supplemented with whole London, UK) at 4°C against glucose-free balanced salt rat serum at concentrations between 10 and 50%, solution (Cockcroft, 1979) for 2 days with two changes of there is an improvement in the growth and differen- dialysis medium. After sterilization dialysed sera were used to culture embryos with the addition of glucose (2mgmr') tiation of embryos as shown by protein content, and vitamin supplement (MEM Vitamin concentrate, morphological score and yolk sac diameter. How- 10/ilmr1). Such sera, both with and without dialysis, were ever, even at 50 % supplementation growth is not supplemented with various concentrations (10-50%) of restored to the levels observed with control serum whole rat serum and the growth of embryos was assessed as (Fig. 1A-C). It has previously been shown that before. In addition 'exhausted' dialysed serum sup- supplementation of Hank's balanced salt solution Serum exhaustion by rat embryos 139

Table 1. Effect of culture in recycled serum on embryonic growth

Morphological Embryonic Yolk sac Yolk sac Passage score protein (jig) diameter (mm) protein (/jg)

1 45 41 -14 ±0-65 206-66 ±6-56 3-5410-06 149-61 15-00 II 50 22-76 ±1-87 84-0415-30 2-91 10-07 72-2313-65 HI 17 3-27 ±0-26 47-1718-90 1-8510-09 29-6413-32 After dialysis - 1V+ 16 17-14±2-19 65-0119-51 2-1110-16 96-4012-59 glucose and vitamins II+glucose 26 39-93 ± 0-49 127-3013-26 3-7410-06 127-1512-07 + vitamins III+glucose 13 12-72 ±2-36 57-1815-21 1-3110-09 53-4415-30 +vitamins After dialysis - IV 11 23-91 ±4-93 88-7318-02 2-8410-33 112-6419-85 +glucose and vitamins Control 20 40-8810-70 205-0017-21 3-5410-06 149-6115-00 Control after dialysis 18 40-33 ± 1-71 149-3316-72 3-1610-41 139-35 17-79 +glucose and vitamins

Results are expressed as the mean 1 S.E.

Table 2. Changes in glucose, lactateand protein levelswith repeated culture Glucose Lactate Protein Passage (mmoir1) (mmoir1) (mgraP1) Fresh serum 8-0910-32 4-6310-39 94-4313-32 I 1-67 + 0-27 14-3710-70 91-7911-14 II 0-3010-05 16-0410-91 97-6512-23 III Not detectable 15-3510-16 94-9711-72 II+glucose 1-6410-17 24-5210-80 83-1814-06 + vitamins III+glucose 4-5110-18 27-2410-31 103-7414-94 +vitamins After dialysis not done 40010-38 not done

Results are expressed as the mean 1 S.E.

Table 3. Effect of lactate on growth indeed growth is completely normal for all para- meters at 30% supplementation (Fig. 2A-C). + Lactate Controls (25 mmoir') Morphological score 43-2512-31 21-4114-40 Effect of repeated culture of embryos on serum ("=11) characteristics Embryonic protein (/ig) 195-0016-62 105-76110-18 Table 2 shows that whilst repeated culture has no effect on the total protein content of serum, the levels of glucose fall dramatically with a concomitant rise in Yolk sac diameter (mm) 3-9210-089 2-9210-19 lactate levels. In order to investigate the effects of high lactate concentrations on the growth of embryos, Results are expressed as the mean 1 S.E. lactate (25 mmol I"1) was added to control serum. At this level, lactate severely inhibited embryonic growth (Table 3). with 50% rat serum permits normal embryonic Repeated culture of embryos in serum causes a growth (Huxham & Beck, 1985), and therefore it is slight drop in the pH of the medium which is not likely that these results are indicative of the presence observed in the absence of embryos. The osmolality of toxic moieties in 'exhausted' serum. If recycled of the medium was found to rise with repeated serum is dialysed prior to supplementation as above, culture, and to a lesser extent in control serum it is found that the restoration of growth occurs at incubated, frozen and thawed in the same way much lower whole rat serum concentrations, and (Table 4). 140 M. K. i^ratten andothers 45 ' A S 45 40 T 4° |35 $ 1 30 i ± s •3 30 1 15 • •a 25 X# "3 20 O20 .c o 1 I"" S *T $ 2 10 * T III r ! [ 10 5 • j.,. 5 ffesjsl] 10 20 30 40 50 C CD ED 10 20 30 40 50

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40 •m txm is:ss C E 10 20 30 40 50 CD ED 10 20 30 40 50 Fig. 1. The effect of supplementation with whole rat serum on the growth-supporting capacity for embryos of Fig. 2. The effect of supplementation with whole rat recycled serum. C, control whole rat serum; E, serum on the growth-supporting capacity for embryos of 'exhausted' serum; 10, 20, 30, 40, 50; percentage of whole dialysed recycled serum. C, control whole rat serum; CD, serum supplementation. Results are the mean of values control dialysed whole rat serum; ED, "exhausted' for at least 20 embryos ± s.E. $ indicates significant dialysed serum; 10, 20, 30, 40, 50. percentage of whole difference from growth in 'exhausted'; serum P<0-01; serum supplementation of dialysed recycled serum. 'indicates significant difference from growth in whole Results are the means of values for at least 20 serum P<0-01 by Students f-test. embryos ± S.E. $ indicates significant difference from growth in 'exhausted' serum P<0-01; * indicates significant difference from growth in whole serum Tables 2 and 4 also show that dialysis restores P < 0-01, # indicates significant difference from growth in lactate, pH and osmolality to levels comparable with 'exhausted' dialysed serum f<0-01 by Students Mest. those found in control serum.

Effect of repeated culture on insulin levels in serum & D'Ercole, 1984; Gluckman. 1986). It was, there- Insulin is known to have growth-promoting proper- fore, of interest to measure the changes in insulin ties for many cell types and fetal tissues (Underwood levels of serum with repeated culture. Table 5 shows Serum exhaustion by rat embryos 141

Table 4. Changes in pH and osmolality in serum 20 A used for repeated culture of embryos T* Osmolality Passage PH (mosmolkg ') io Fresh serum 7-88 ± 0-08 326-0 ± 150 Cultured without Is embryos I 7-90 ±0-10 323-0 ± 6-0 II 7-91 ±0-09 348-0+ 11-2 0 5 10 15 20 25 III 7-80 ±0-13 407-8 ± 11-6 Insulin concentration (ng ml ') Cultured with embryos I 7-28 ±0-12 341-1 ±10-1 II 7-29 ± 0-08 399-6 ±5-5 III 7-23 ±0-13 427-0 ±15-2 2 After dialysis 7-93 ±0-01 295-0 ±0-1 fa Results are expressed as the mean± S.E. a I Table 5. Effect of repeated culture on serum insulin levels Passage Insulin (ngml ')

Fresh serum 1-25 ±019 0 5 10 15 20 25 I 1-00 ±0-18 Insulin concentration (ngmr ') II 0-73 ±0-22 80 II + glucose 0-71 ±0-22 +vitamins 70 III + glucose 0-49 ± 0-05 1* +vitamins

Results are expressed as the mean ± S.E. §40 .5 30 u that the insulin levels fall with repeated culture, o 20 suggesting that hormones and related molecules may °- 10 0 be specifically depleted by the embryos under such 0 5 10 15 20 25 culture conditions. Insulin concentration (ngml ') Effect of supplementation of recycled dialysed serum Fig. 3. The effect of supplementation with porcine insulin with specific growth-promoting factors on the growth-supporting capacity of dialysed recycled rat Addition of porcine insulin at concentrations between serum for embryos cultured in vitro. The x axis shows the 5 and 25ngml~' slightly improved the growth and final concentration of the insulin supplement. Results are differentiation of embryos grown in dialysed 'exhaus- the mean of values for at least 15 embryos ± S.E. ted' serum supplemented with glucose and vitamins, * Indicates significant difference from growth in although there was no significant improvement above 'exhausted' dialysed serum P<0-01 by Students f-test. lOngml"1 (Fig. 3A-C). At concentrations above 1 50ngml" the addition of insulin had a detrimental Addition of rat transferrin at 500 fig ml ' caused an effect (results not shown). When insulin was added to improvement in embryonic growth (Fig. 5). In ad- whole rat serum at concentrations less than 1 dition, the embryos were observed to have numerous, 50ngml" it was without effect, whereas concen- apparently normal, red blood cells in the circulation, trations above this level proved to be embryotoxic. whereas in those cultured in unsupplemented sera, When epidermal growth factor was added to dia- when the circulation was sufficiently well developed lysed recycled serum, an improvement in the growth for this to be assessed, the blood cells were colour- and differentiation of cultured embryos was observed less. The other factors (with the exception of whole with increasing concentrations over the range serum supplementation) added to recycled serum did 5-25ngml"1 (Fig. 4A-C). not have an effect on the overt anaemia. 142 M. K. Pratten and others

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Fig. 5. The effect of supplementation with transferrin (500//gml"1) on the growth-supporting capacity of dialysed recycled rat serum for embryos cultured in vitro. Results are the mean of values for at least 15 embryos ± S.E. * Indicates significant difference from growth in 'exhausted' dialysed serum P<0-01 by

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agreement with the work of New et al. (1976), Kleiner al. (1978) and Sanyal (1980). It is possible to suggest 0 5 10 15 25 several reasons for this. One explanation is that when EGF concentration (ng ml ') serum is used in this way the repeated freezing, 50 c thawing and filtration of the serum renders it in- 45 capable of supporting normal growth and differen- _40 tiation of embryos. However, when serum is treated 1 • in exactly the same way but no embryos are cultured in H in it, it is still able to support normal embryonic 5 30- m development. There are two other explanations for § 25 • [ this effect; one is that the growth of embryos in serum S 20 adds factors to serum, possibly waste products, which u 15 I render it toxic to further embryos; the other is that the embryos deplete material from the serum, poss- 10 p ' h ibly proteins or hormones, which makes further 5 !.mil. L 1. embryonic growth impossible. This study goes some 0 0 5 10 15 25 EGF concentration (ngml ') way to elucidating the mechanism by which serum is 'exhausted'. Fig. 4. The effect of supplementation with epidermal Our data indicate that as glucose in serum is used growth factor on the growth-supporting capacity of by embryos there is a build up of lactate (Table 2). dialysed recycled rat serum for embryos cultured in vitro. Since other workers have shown that at this stage of The x axis shows the final concentration of the epidermal gestation the embryonic is based primar- growth factor supplement. Results are the mean of values ily on (Tanimura & Shepherd, 1970), it is for at least 15 embryos ± S.E. * Indicates significant difference from growth in 'exhausted' dialysed serum likely that lactate is the major breakdown product of P<0-01 by Students Mest. carbohydrate metabolism. Although we have shown that lactate itself very severely inhibits embryonic growth when present at a concentration of Discussion 25mmoll~' (Table 3), at a level of around 15mmoll~', as found at the end of a single embryo Embryonic growth is clearly poorer in recycled serum culture (Table 2), the effect is far less marked than in fresh serum (Table 1). This is in general providing the glucose levels are restored to normal. Serum exhaustion by rat embryos 143

This is shown by results obtained following the rin. Previous work (Calvert et al. 1986; Andrews et al. second passage plus glucose and vitamins (Table 1). 1987; Cumberland et al. 1987) has suggested a role for However, when lactate is removed by dialysis of these factors in the growth and development of rat serum following the third passage, then glucose is embryos in vitro. added, very poor growth of embryos is obtained. The Insulin improved growth and development with a other potentially harmful waste products such as maximum effect at around lOngml"1, which is close urea, uric acid and creatinine (Sanyal, 1980) are of to the physiological level. These results are interest- similar molecular weight to lactate and are therefore ing in regard to the recent data indicating that also likely to be removed by dialysis. It is possible, normoglycaemic serum, specifically depleted of insu- however, that some nondialysable toxic agents are lin using an affinity column, is not able to support formed during embryos culture and thus inhibit normal growth and development, whilst this effect further embryonic growth. can be reversed by addition of insulin at lOngmr1 When whole rat serum was used to supplement (Travers & Beck, 1988). 'exhausted' serum, it was not possible to restore its The role of insulin and insulin-like growth factors growth-promoting properties even when it was sup- (IGFs) in the regulation of fetal growth is fairly well plemented at the 50% level (Fig. 1). When Hank's established (Gluckman, 1986), but the role of these balanced salt solution and whole rat serum are used in factors at earlier stages of gestation is less well a 1:1 (v/v) ratio, normal growth ensues (Huxham & understood. The for insulin has been ident- Beck, 1985). This is further evidence that one of the ified on both embryonic tissues and extraembryonic causes of the lack of growth of embryos in 'exhausted' membranes for rat conceptuses at gestational ages 10 serum is the presence of toxic metabolites. and 12 days (Unterman etal. 1986), and recent studies The removal of low molecular weight toxic ma- have identified receptors for IGF-I and IGF-II, as terials by mild dialysis has the effect of permitting well as insulin, in mouse embryos at the stage of normal embryonic growth when 30 % whole serum organogenesis (Smith etal. 1987). It has been known supplementation is employed. 30% levels of whole for some time that the major fetal in serum supplementation are insufficient to support both and rats is IGF-II, and also that recep- normal embryonic development when added to tors for both IGF-I and IGF-II are present in fetal balanced salt solution only. It is clear therefore that tissues (Sara & Hall, 1984). With the advent of in situ low molecular weight toxic metabolites are released hybridization histochemistry, it has been possible to into the serum as it is recycled for culture of embryos, demonstrate recently the presence of mRNA for but since the adverse effect can be removed by IGF-II in both human (Han etal. 1987) and rat (Beck dialysis, the presence of other higher molecular et al. 1987) embryonic tissues as well, although only weight toxic materials is excluded. However, since low levels were present during the early stages of normal growth can only be obtained when such organogenesis, as studied here. In the rat study it was dialysed recycled sera are supplemented with whole also shown that very little IGF-I is produced by the rat serum, it is probable that some growth-promoting conceptus at early gestational ages. The earliest materials, which are normally present in serum at low identification of insulin in rat embryonic by levels, are depleted during repeated culture so that immunocytochemistry was at 13-5 days of gestation they become growth limiting. Many growth factors (Travers & Beck, 1988), which suggests that any and hormones fall into this category. insulin requirement by the embryo at earlier stages Other workers (Klein et al. 1978; Priscott et al. must be provided from maternal sources. When 1983) have suggested that certain specific proteins, embryos are grown in vitro the source of exogenous including transferrin, a2 macroglobulin, at lipopro- factors is the serum culture medium. tein and ceruloplasmin, and also some uncharacter- When epidermal growth factor was added to serum ized proteins of 125 and >200xl03 relative molecular depleted of factors by repeated use for the culture of mass are depleted from serum by embryo culture. rat embryos, it was found to improve the growth of Although we present no comprehensive evidence for further embryos in a dose-dependent manner. The this, it is certainly the case that insulin levels drop concentrations at which a plateauing of this effect was quite markedly with repeated culture (Table 5). It has observed (15-25 ng ml"1) are in the physiological recently been shown that extremely low insulin levels range. It has been proposed that effects observed at found in serum specifically depleted by column such concentrations of are likely to be by chromatography preclude normal embryonic growth interactions with a highly specific receptor (Gospo- in culture, the level for this effect to be apparent dorowicz, 1981; Heath & Rees, 1985; Pratt, 1983). being less than 0-5ngmr' (Travers & Beck, 1988). The presence of an EGF receptor on fetal mouse and Three specific factors have been chosen for investi- rat tissues has been reported (Hortsch et al. 1983), gation: insulin, epidermal growth factor and transfer- although the same study failed to identify receptors at 144 M. K. Pratten and others early stages, especially in the yolk sac. Earlier studies 1984; Rizzino, 1987) and it is likely that the factors (Nexo et al. 1980) had shown the presence of both added in this study are normally provided by the receptors for EGF and an endogenous growth factor, maternal system. Whilst evidence is now accumulat- later identified as transforming growth factor a ing regarding the synthetic capability of the embry- (TGFa"). Mouse embryonic tissues have been demon- onic tissues for growth factors (e.g. Williams et al. strated to contain TGFar (Twardzik, 1982) which acts 1986; Han etal. 1987; Beck et al. 1987), in most cases via the EGF receptor. It is, however, possible that synthesis is only at a significant level in the late exogenous EGF is required before the establishment organogenetic period and it is possible that the early of EGF or TGFar synthesis within the embryo (Riz- postimplantation embryo, as studied here, is depen- zino, 1987) and that, in the model used here, this is a dent on maternal factors for the regulation of its factor which becomes depleted from serum by recyc- growth and differentiation. ling. Transferrin had two effects when added to recycled Thanks are due to the Wellcome Trust and the Medical serum; first it improved the growth of the embryos, Research Council for grants in aid of this research. The and second it averted the anaemia observed with skilled technical assistance of Timothy Jefferson is also unsupplemented serum. The latter effect is not sur- gratefully acknowledged. prising since transferrin has a major role in iron transport into cells, as well as being essential for cell References viability (Morgan et al. 1978) and is therefore likely to have effects on haemoglobin synthesis. In addition, it ADAMSON, E. D. (1983). Growth factors in development. has previously been reported that the level of trans- In The Biological Basis of Reproductive and ferrin in serum is decreased by embryonic culture Developmental Medicine (ed. J. B. Warshaw), (Priscott et al. 1983). There have been reports that pp. 307-336. London: Edward Arnold. transferrin is synthesized by the visceral yolk sac ANDREWS, K. E., PRATTEN, M. K. & BECK, F. (1987). (Williams et al. 1986) but it is clear that a large Epidermal growth factor: pinocytosis and effect on proportion of the embryonic transferrin requirement embryonic development. Biochem. Soc. Trans. 15, in the postimplantation conceptus is of maternal 920-921. origin and is transported across the yolk sac BECK, F., SAMANI, N. J., PENSHOW, J. D., THORLEY, B., (Huxham & Beck, 1985; Cumberland et al. 1987). TREGEAR, G. W. & COGHLAN, J. P. (1987). Histochemical localization of IGF-I and -II mRNA in The improved growth and differentiation of em- the developing rat embryo. Development 101, 175-184. bryos in recycled serum in the presence of exogenous BROCK, J. H., MAINOU-FOWLER, T. & WEBSTER, L. M. rat transferrin may be entirely due to its iron- (1986). Evidence that transferrin may function transporting role, since many systems in cells exclusively as an iron donor in promoting lymphocyte require iron or other metals for their activity. How- proliferation. Immunology 57, 105-110. ever, it has been suggested that transferrin and its BROWN, N. A. & FABRO, S. (1981). Quantitation of rat receptor may have a growth-promoting role for embryonic development in vitro: A morphological cultured cells independent of any iron-transport func- scoring system. Teratology 224, 65-78. tion. Studies performed using alternative sources of CALVERT, N. I. R., PRATTEN, M. K. & BECK, F. (1986). iron carrier for the cells or occupancy of the receptor Trophic factors in rat serum and embryonic by antibodies against the receptor have attempted to development. Biochem. Soc. Trans. 14, 980—981. COCKCROFT, D. L. (1979). Nutrient requirements of rat separate the iron-transport function from other po- embryos undergoing organogenesis in vitro. J. Reprod. tential intracellular signalling events which may occur Fen. 57, 505-510. during receptor occupancy and internalization (Ek- COCKCROFT, D. L. & COPPOLA, P. T. (1977). Teratogenic blom & Thesleff, 1985; Brock et al. 1986; Landschulz effects of excess glucose on head-fold rat embryos in et al. 1984; and Beck et al. 1987). It remains unclear culture. Teratology 16, 141-146. whether or not there are two independent actions of CUMBERLAND, P. F. T., MENSAH-BROWN, E. P. K., transferrin in this context, and whether or not this MURAKAMI, T. & PRATTEN, M. K. (1987). Species- applies to the effect on embryonic growth is difficult specificity of transferrin in embryonic growth. Biochem. to ascertain. Soc. Trans. 15, 919-920. Not surprisingly, in no case was it possible to DUNTON, A., AL-ALOUSI, L., PRATTEN, M. K. & BECK, F. restore growth and differentiation of rat embryos to (1986). The giant yolk sac; a model for studying early placental transport. /. Anat. 145, 189-206. the level observed when whole rat serum supplemen- EKBLOM, P. & THESLEFF, I. (1985). Control of tation was used by the addition of a single growth- differentiation by soluble factors secreted by the promoting factor. However, several reviews discuss a embryonic and yolk sac. Devi Biol. 110, 29-38. role for such molecules in embryonic and fetal devel- FREEMAN, S. J., BECK, F. & LLOYD, J. B. (1981). The role opment (e.g. Adamson, 1983; Heldin & Westermark, of the visceral yolk sac in mediating protein utilisation Serum exhaustion by rat embryos 145

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