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

Transcription

1 Development 4, (1988) Printed in Great Britain The Company of Biologists Limited The effet of epidermal growth fator, insulin and transferrin on the growth-promoting properties of serum depleted by repeated ulture of postimplantation rat embryos M. K. PRATTEN, A. M. BROOKE, S. C. BROOME and F. BECK Department of Anatomy, University of Leiester, Leiester LE 7RH, UK Summary Homologous serum, when repeatedly used for the ulture of postimplantation rat embryos, rapidly loses its apaity to support growth and development. Replenishment of the 'exhausted' serum with gluose and vitamins (MEM vitamin onentrate - Flow Laboratories) together with gentle dialysis to remove small moleular weight toxi metabolites (latate et) fails to restore the growth-promoting properties of the serum. This suggests that 'reyled' serum has been depleted of speifi growth-promoting fators. Suh serum that has been subjeted to dialysis an be ompletely replenished by addition of 3 % normal rat serum. t is therefore probable that the growth promoters are originally present at very low onentrations and beome rate limiting when serum is reyled. Many growth fators and hormones fall into this ategory and it is likely that a onsiderable number are involved when serum is 'exhausted' by repeated use. When insulin, epidermal growth fator or rat transferrin are added to dialysed 'exhausted' serum eah effets a partial restoration of growth of rat embryos. Key words: rat embryo ulture, reyled serum, growth of embryos, growth fators. ntrodution Although it has been apparent for some time that homologous serum has a finite growth-supporting apaity for postimplantation rat embryos ultured in vitro, the important biohemial fators involved have not been effetively identified. Some dilution of the serum is possible whilst still maintaining normal growth (Cokroft & Coppola, 1977; Huxham & Bek, 198) but the studies performed indiate that % serum is required for growth to be omparable to that observed in utero. When the rat serum is replaed by human serum, even with the addition of exess gluose, growth of rat embryos is not ompletely normal unless a small proportion ( %) of rat serum is added (Gupta & Bek, 1983; Lear et al. 1983). This implies that there are speies-speifi fators in rat serum whih the embryo requires. The nutritional role of the rat viseral yolk sa is well established (Freeman et al. 1981). n addition, it is now lear that the yolk sa transports many maromoleules to the embryo intat (Huxham & Bek, 1984, 198). ndeed, the fat that many suh fators are of importane to embryoni growth is demonstrated by the evidene that fluid ontained within the ultured viseral yolk sa has some growthpromoting properties for rat embryos (Dunton et al. 1986). t has been suggested by Klein et al. (1978) that ertain speifi proteins are depleted from serum when rat embryos are ultured at high density. Also Sanyal (198) has shown that rat embryos mediate hanges in the omponents of the ulture medium, partiularly in terms of ph, Po> gluose, urea and reatinine. More reently Prisott (Prisott et al. 1983) has to some degree extended the studies on serum proteins and has tentatively identified the proteins that are depleted by rat embryos in ulture as a^-maroglobulin and transferrin, amongst others. n this study, we have attempted to 'exhaust' the growth-supporting apaity of rat serum by using it for repeated ulture. We have assessed the extent of embryoni growth and development with reyled serum and have attempted to eluidate whether the

2 138 M. K. Pratten and others limited growth observed was due to depletion of speifi fators or to toxi hanges in the serum brought about by the repeated ulture. n addition, the serum has been supplemented with fators that may be depleted during repeated ulture in an attempt to identify some of the speifi agents involved. Materials and methods Culture of rat embryos in whole and reyled rat serum Wistar rats were mated overnight and pregnany was timed from midnight preeding the morning on whih vaginal plugs were observed. Coneptuses were explanted at 9- days (early head-fold stage, ontaining three somites), aording to the standard method of rodent embryo ulture desribed by New (1978). Briefly, explanted embryos were ultured in homologous sera (see below) at 37 C in glass bottles in a roller inubator. At the start of ulture the inubation bottles were gassed with % O 2 : % CO 2 : 9 % N 2, after 24 h with 2% O 2 : % CO 2 : 7 % N 2 and after 44 h with 4 % O 2 : % CO 2 : % N 2. Serum was prepared from both male and female rats by immediate entrifugation and stored at 18 C until used. Before ulture the serum was heat-inativated at 6 C for 3min. From a pool of approximately ml serum, ml was reserved as ontrol serum and was treated in exatly the same way as experimental serum exept that it was ultured without embryos. n this way, the effets of inubation, freezing and thawing and filtration were eliminated. The remainder was used as ulture medium for embryos from days of development at a density of 1 embryo ml"'. After ulture for h the serum was frozen and stored. t was then sterilized by passage through a Millex-GV -22 urn filter unit (Millipore R ) and reused for ulture of further embryos with or without the addition of 2mgml~' gluose and ^1 ml" 1 MEM vitamin onentrate (Flow Laboratories). A small sample of medium was removed at eah stage for biohemial analysis. Serum was reyled in this way until the ulture of explants resulted in no signifiant growth as assessed by the morphologial soring system of Brown & Fabro (1981) whih assesses the extent of growth and differentiation of 13 different embryologial features inluding development of neural tube, limb bud, heart, oti and opti systems and yolk sa. n addition, measurements were made of protein levels (Lowry et al. 191), yolk sa diameter, rown-rump length and somite number. Suh serum was onsidered to be 'exhausted'. 'Exhausted' serum and whole serum were then dialysed using Visking size 2 tubing (Mediell nternational Ltd, London, UK) at 4 C against gluose-free balaned salt solution (Cokroft, 1979) for 2 days with two hanges of dialysis medium. After sterilization dialysed sera were used to ulture embryos with the addition of gluose (2mgmr') and vitamin supplement (MEM Vitamin onentrate, /ilmr 1 ). Suh sera, both with and without dialysis, were supplemented with various onentrations (-%) of whole rat serum and the growth of embryos was assessed as before. n addition 'exhausted' dialysed serum supplemented with gluose and vitamins was also used with the addition of insulin (-2 ng ml" 1 ), epidermal growth fator (-2 ngml"') or rat transferrin. The extent of growth and differentiation of embryos was assessed after a total of h in ulture as above. Epidermal growth fator (from mouse submaxillary gland) and insulin (porine, rystalline, - % Zn) were obtained from Sigma Chemial Co. Ltd, Poole, Dorset, UK. Rat transferrin was prepared by the method of Shreiber et al. (1979). Biohemial methods The ph of samples of sera were measured with a Pye Uniam digital ph meter. Care was taken to keep samples in airtight ontainers before measurements were performed to avoid any hanges in ph. The osmolality was measured using a Halbmikro osmometer. Latate levels were measured using a diagnosti kit from Sigma Chemial Co., gluose levels were measured by the gluose oxidase method (kit from Sigma Chemial Co.) and total protein was assessed by a modifiation of the Lowry method (Lowry et al. 191). The levels of insulin in fresh and reyled sera were assayed using a double antibody radiolabelling method (Wellome Laboratories). Results Effet of ulture in reyled serum on embryoni growth Table 1 shows that the growth of embryos in serum that has been reyled one is greatly retarded. f the serum was used again hardly any embryoni growth was obtained. Even when gluose (2mgml~') and vitamins (jdml"' minimum essential medium onentrate) were added bak to reyled serum, embryoni growth was severely impaired. Control embryos grown in serum subjeted to the same regime of freezing and thawing grew normally. When reyled serum was dialysed (to remove any low moleular weight toxi waste produts) and gluose and vitamins added, only limited improvement in growth was observed, indiating that the loss of growth-supporting properties an only be partially reversed in this way. After similar dialysis of whole serum embryoni growth was hardly affeted in terms of morphologial sore or yolk sa diameter, although there was some effet on the protein level of the embryos. When reyled serum is supplemented with whole rat serum at onentrations between and %, there is an improvement in the growth and differentiation of embryos as shown by protein ontent, morphologial sore and yolk sa diameter. However, even at % supplementation growth is not restored to the levels observed with ontrol serum (Fig. 1A-C). t has previously been shown that supplementation of Hank's balaned salt solution

3 Table 1. Effet of ulture in reyled serum on embryoni growth Serum exhaustion by rat embryos 139 Passage Morphologial sore Embryoni protein (jig) Yolk sa diameter (mm) Yolk sa protein (/jg) 1 H After dialysis - 1V+ gluose and vitamins ± ± ± ± ± gluose + vitamins +gluose +vitamins After dialysis - V +gluose and vitamins ± ± ± Control Control after dialysis +gluose and vitamins ± Results are expressed as the mean 1 S.E. Table 2. Changes in gluose, latateand protein levelswith repeated ulture Passage Gluose (mmoir 1 ) Latate (mmoir 1 ) Protein (mgrap 1 ) Fresh serum Not detetable gluose vitamins +gluose vitamins After dialysis not done 4-38 not done Results are expressed as the mean 1 S.E. Table 3. Effet of latate on growth + Latate Morphologial sore Controls (2 mmoir') ("=11) Embryoni protein (/ig) Yolk sa diameter (mm) Results are expressed as the mean 1 S.E. with % rat serum permits normal embryoni growth (Huxham & Bek, 198), and therefore it is likely that these results are indiative of the presene of toxi moieties in 'exhausted' serum. f reyled serum is dialysed prior to supplementation as above, it is found that the restoration of growth ours at muh lower whole rat serum onentrations, and indeed growth is ompletely normal for all parameters at 3% supplementation (Fig. 2A-C). Effet of repeated ulture of embryos on serum harateristis Table 2 shows that whilst repeated ulture has no effet on the total protein ontent of serum, the levels of gluose fall dramatially with a onomitant rise in latate levels. n order to investigate the effets of high latate onentrations on the growth of embryos, latate (2 mmol " 1 ) was added to ontrol serum. At this level, latate severely inhibited embryoni growth (Table 3). Repeated ulture of embryos in serum auses a slight drop in the ph of the medium whih is not observed in the absene of embryos. The osmolality of the medium was found to rise with repeated ulture, and to a lesser extent in ontrol serum inubated, frozen and thawed in the same way (Table 4).

4 14 M. K. i ^ratten andothers 4 ' A 4 4 S "3 2. o 2 T $ 3 *T f] i ±s 1! [ r O2 "" S *T $ j.,. fesjsl X# a 2 C CD ED r B i3 \ # 2 24r CD ED $ 2 16 i : J* o SO a. 4 EL. E 4 C E 2 m txm is:ss 3 4 Fig. 1. The effet of supplementation with whole rat serum on the growth-supporting apaity for embryos of reyled serum. C, ontrol whole rat serum; E, 'exhausted' serum;, 2, 3, 4, ; perentage of whole serum supplementation. Results are the mean of values for at least 2 embryos ± s.e. $ indiates signifiant differene from growth in 'exhausted'; serum P<-1; 'indiates signifiant differene from growth in whole serum P<-1 by Students f-test. Tables 2 and 4 also show that dialysis restores latate, ph and osmolality to levels omparable with those found in ontrol serum. Effet of repeated ulture on insulin levels in serum nsulin is known to have growth-promoting properties for many ell types and fetal tissues (Underwood CD ED Fig. 2. The effet of supplementation with whole rat serum on the growth-supporting apaity for embryos of dialysed reyled serum. C, ontrol whole rat serum; CD, ontrol dialysed whole rat serum; ED, "exhausted' dialysed serum;, 2, 3, 4,. perentage of whole serum supplementation of dialysed reyled serum. Results are the means of values for at least 2 embryos ± S.E. $ indiates signifiant differene from growth in 'exhausted' serum P<-1; * indiates signifiant differene from growth in whole serum P < -1, # indiates signifiant differene from growth in 'exhausted' dialysed serum f<-1 by Students Mest. & D'Erole, 1984; Glukman. 1986). t was, therefore, of interest to measure the hanges in insulin levels of serum with repeated ulture. Table shows

5 Serum exhaustion by rat embryos 2 Table 4. Changes in ph and osmolality in serum used for repeated ulture of embryos Passage Fresh serum PH Osmolality (mosmolkg ') 7-88 ± ± A T* io s Cultured without embryos 7-9 ± ± ± ± ± 11-6 Cultured with embryos 7-28 ± ± ± ± ± ±1-2 After dialysis 7-93 ± ± nsulin onentration (ng ml ') nsulin onentration (ngmr ') nsulin onentration (ngml ') 2 fa2 Results are expressed as the mean± S.E. a Table. Effet of repeated ulture on serum insulin levels Passage Fresh serum + gluose +vitamins + gluose +vitamins nsulin (ngml ') 1-2 ±19 1- ± ± ± ± - Results are expressed as the mean ± S.E * 4. 3 u that the insulin levels fall with repeated ulture, suggesting that hormones and related moleules may be speifially depleted by the embryos under suh ulture onditions. Effet of supplementation of reyled dialysed serum with speifi growth-promoting fators Addition of porine insulin at onentrations between and 2ngml~' slightly improved the growth and differentiation of embryos grown in dialysed 'exhausted' serum supplemented with gluose and vitamins, although there was no signifiant improvement above longml"1 (Fig. 3A-C). At onentrations above ngml"1 the addition of insulin had a detrimental effet (results not shown). When insulin was added to whole rat serum at onentrations less than ngml" 1 it was without effet, whereas onentrations above this level proved to be embryotoxi. When epidermal growth fator was added to dialysed reyled serum, an improvement in the growth and differentiation of ultured embryos was observed with inreasing onentrations over the range -2ngml" 1 (Fig. 4A-C). o 2 - Fig. 3. The effet of supplementation with porine insulin on the growth-supporting apaity of dialysed reyled rat serum for embryos ultured in vitro. The x axis shows the final onentration of the insulin supplement. Results are the mean of values for at least 1 embryos ± S.E. * ndiates signifiant differene from growth in 'exhausted' dialysed serum P<-1 by Students f-test. Addition of rat transferrin at fig ml ' aused an improvement in embryoni growth (Fig. ). n addition, the embryos were observed to have numerous, apparently normal, red blood ells in the irulation, whereas in those ultured in unsupplemented sera, when the irulation was suffiiently well developed for this to be assessed, the blood ells were olourless. The other fators (with the exeption of whole serum supplementation) added to reyled serum did not have an effet on the overt anaemia.

6 142 M. K. Pratten and others 3 A " S2 3 A B 6 r C T± o ^' "So 34 2 \? o a is T " 1 2 EGF onentration (ngml"1) Q (j 3 -** 1 T 2 -> ^ Rat transferhn Rat transferrin Rat transferrin onentration (/<gml ') onentration (jjgml ') onentration Fig.. The effet of supplementation with transferrin (//gml"1) on the growth-supporting apaity of dialysed reyled rat serum for embryos ultured in vitro. Results are the mean of values for at least 1 embryos ± S.E. * ndiates signifiant differene from growth in 'exhausted' dialysed serum P<-1 by Students Mest EGF onentration (ng ml ') 4 _4 1in H m 3 2 S 2 [ 1 u p ' h L EGF onentration (ngml ')!.mil. Fig. 4. The effet of supplementation with epidermal growth fator on the growth-supporting apaity of dialysed reyled rat serum for embryos ultured in vitro. The x axis shows the final onentration of the epidermal growth fator supplement. Results are the mean of values for at least 1 embryos ± S.E. * ndiates signifiant differene from growth in 'exhausted' dialysed serum P<-1 by Students Mest. Disussion Embryoni growth is learly poorer in reyled serum than in fresh serum (Table 1). This is in general agreement with the work of New et al. (1976), Kleiner al. (1978) and Sanyal (198). t is possible to suggest several reasons for this. One explanation is that when serum is used in this way the repeated freezing, thawing and filtration of the serum renders it inapable of supporting normal growth and differentiation of embryos. However, when serum is treated in exatly the same way but no embryos are ultured in it, it is still able to support normal embryoni development. There are two other explanations for this effet; one is that the growth of embryos in serum adds fators to serum, possibly waste produts, whih render it toxi to further embryos; the other is that the embryos deplete material from the serum, possibly proteins or hormones, whih makes further embryoni growth impossible. This study goes some way to eluidating the mehanism by whih serum is 'exhausted'. Our data indiate that as gluose in serum is used by embryos there is a build up of latate (Table 2). Sine other workers have shown that at this stage of gestation the embryoni metabolism is based primarily on glyolysis (Tanimura & Shepherd, 197), it is likely that latate is the major breakdown produt of arbohydrate metabolism. Although we have shown that latate itself very severely inhibits embryoni growth when present at a onentration of 2mmoll~' (Table 3), at a level of around 1mmoll~', as found at the end of a single embryo ulture (Table 2), the effet is far less marked providing the gluose levels are restored to normal.

7 Serum exhaustion by rat embryos 143 This is shown by results obtained following the seond passage plus gluose and vitamins (Table 1). However, when latate is removed by dialysis of serum following the third passage, then gluose is added, very poor growth of embryos is obtained. The other potentially harmful waste produts suh as urea, uri aid and reatinine (Sanyal, 198) are of similar moleular weight to latate and are therefore also likely to be removed by dialysis. t is possible, however, that some nondialysable toxi agents are formed during embryos ulture and thus inhibit further embryoni growth. When whole rat serum was used to supplement 'exhausted' serum, it was not possible to restore its growth-promoting properties even when it was supplemented at the % level (Fig. 1). When Hank's balaned salt solution and whole rat serum are used in a 1:1 (v/v) ratio, normal growth ensues (Huxham & Bek, 198). This is further evidene that one of the auses of the lak of growth of embryos in 'exhausted' serum is the presene of toxi metabolites. The removal of low moleular weight toxi materials by mild dialysis has the effet of permitting normal embryoni growth when 3 % whole serum supplementation is employed. 3% levels of whole serum supplementation are insuffiient to support normal embryoni development when added to balaned salt solution only. t is lear therefore that low moleular weight toxi metabolites are released into the serum as it is reyled for ulture of embryos, but sine the adverse effet an be removed by dialysis, the presene of other higher moleular weight toxi materials is exluded. However, sine normal growth an only be obtained when suh dialysed reyled sera are supplemented with whole rat serum, it is probable that some growth-promoting materials, whih are normally present in serum at low levels, are depleted during repeated ulture so that they beome growth limiting. Many growth fators and hormones fall into this ategory. Other workers (Klein et al. 1978; Prisott et al. 1983) have suggested that ertain speifi proteins, inluding transferrin, a 2 maroglobulin, a t lipoprotein and eruloplasmin, and also some unharaterized proteins of 12 and >2xl 3 relative moleular mass are depleted from serum by embryo ulture. Although we present no omprehensive evidene for this, it is ertainly the ase that insulin levels drop quite markedly with repeated ulture (Table ). t has reently been shown that extremely low insulin levels found in serum speifially depleted by olumn hromatography prelude normal embryoni growth in ulture, the level for this effet to be apparent being less than -ngmr' (Travers & Bek, 1988). Three speifi fators have been hosen for investigation: insulin, epidermal growth fator and transferrin. Previous work (Calvert et al. 1986; Andrews et al. 1987; Cumberland et al. 1987) has suggested a role for these fators in the growth and development of rat embryos in vitro. nsulin improved growth and development with a maximum effet at around longml" 1, whih is lose to the physiologial level. These results are interesting in regard to the reent data indiating that normoglyaemi serum, speifially depleted of insulin using an affinity olumn, is not able to support normal growth and development, whilst this effet an be reversed by addition of insulin at longmr 1 (Travers & Bek, 1988). The role of insulin and insulin-like growth fators (GFs) in the regulation of fetal growth is fairly well established (Glukman, 1986), but the role of these fators at earlier stages of gestation is less well understood. The reeptor for insulin has been identified on both embryoni tissues and extraembryoni membranes for rat oneptuses at gestational ages and 12 days (Unterman etal. 1986), and reent studies have identified reeptors for GF- and GF-, as well as insulin, in mouse embryos at the stage of organogenesis (Smith etal. 1987). t has been known for some time that the major fetal somatomedin in both humans and rats is GF-, and also that reeptors for both GF- and GF- are present in fetal tissues (Sara & Hall, 1984). With the advent of in situ hybridization histohemistry, it has been possible to demonstrate reently the presene of mrna for GF- in both human (Han etal. 1987) and rat (Bek et al. 1987) embryoni tissues as well, although only low levels were present during the early stages of organogenesis, as studied here. n the rat study it was also shown that very little GF- is produed by the oneptus at early gestational ages. The earliest identifiation of insulin in rat embryoni panreas by immunoytohemistry was at 13- days of gestation (Travers & Bek, 1988), whih suggests that any insulin requirement by the embryo at earlier stages must be provided from maternal soures. When embryos are grown in vitro the soure of exogenous fators is the serum ulture medium. When epidermal growth fator was added to serum depleted of fators by repeated use for the ulture of rat embryos, it was found to improve the growth of further embryos in a dose-dependent manner. The onentrations at whih a plateauing of this effet was observed (1-2 ng ml" 1 ) are in the physiologial range. t has been proposed that effets observed at suh onentrations of ligand are likely to be by interations with a highly speifi reeptor (Gospodorowiz, 1981; Heath & Rees, 198; Pratt, 1983). The presene of an EGF reeptor on fetal mouse and rat tissues has been reported (Hortsh et al. 1983), although the same study failed to identify reeptors at

8 144 M. K. Pratten and others early stages, espeially in the yolk sa. Earlier studies (Nexo et al. 198) had shown the presene of both reeptors for EGF and an endogenous growth fator, later identified as transforming growth fator a (TGFa"). Mouse embryoni tissues have been demonstrated to ontain TGFar (Twardzik, 1982) whih ats via the EGF reeptor. t is, however, possible that exogenous EGF is required before the establishment of EGF or TGFar synthesis within the embryo (Rizzino, 1987) and that, in the model used here, this is a fator whih beomes depleted from serum by reyling. Transferrin had two effets when added to reyled serum; first it improved the growth of the embryos, and seond it averted the anaemia observed with unsupplemented serum. The latter effet is not surprising sine transferrin has a major role in iron transport into ells, as well as being essential for ell viability (Morgan et al. 1978) and is therefore likely to have effets on haemoglobin synthesis. n addition, it has previously been reported that the level of transferrin in serum is dereased by embryoni ulture (Prisott et al. 1983). There have been reports that transferrin is synthesized by the viseral yolk sa (Williams et al. 1986) but it is lear that a large proportion of the embryoni transferrin requirement in the postimplantation oneptus is of maternal origin and is transported aross the yolk sa plaenta (Huxham & Bek, 198; Cumberland et al. 1987). The improved growth and differentiation of embryos in reyled serum in the presene of exogenous rat transferrin may be entirely due to its irontransporting role, sine many enzyme systems in ells require iron or other metals for their ativity. However, it has been suggested that transferrin and its reeptor may have a growth-promoting role for ultured ells independent of any iron-transport funtion. Studies performed using alternative soures of iron arrier for the ells or oupany of the reeptor by antibodies against the reeptor have attempted to separate the iron-transport funtion from other potential intraellular signalling events whih may our during reeptor oupany and internalization (Ekblom & Thesleff, 198; Brok et al. 1986; Landshulz et al. 1984; and Bek et al. 1987). t remains unlear whether or not there are two independent ations of transferrin in this ontext, and whether or not this applies to the effet on embryoni growth is diffiult to asertain. Not surprisingly, in no ase was it possible to restore growth and differentiation of rat embryos to the level observed when whole rat serum supplementation was used by the addition of a single growthpromoting fator. However, several reviews disuss a role for suh moleules in embryoni and fetal development (e.g. Adamson, 1983; Heldin & Westermark, 1984; Rizzino, 1987) and it is likely that the fators added in this study are normally provided by the maternal system. Whilst evidene is now aumulating regarding the syntheti apability of the embryoni tissues for growth fators (e.g. Williams et al. 1986; Han etal. 1987; Bek et al. 1987), in most ases synthesis is only at a signifiant level in the late organogeneti period and it is possible that the early postimplantation embryo, as studied here, is dependent on maternal fators for the regulation of its growth and differentiation. Thanks are due to the Wellome Trust and the Medial Researh Counil for grants in aid of this researh. The skilled tehnial assistane of Timothy Jefferson is also gratefully aknowledged. Referenes ADAMSON, E. D. (1983). Growth fators in development. n The Biologial Basis of Reprodutive and Developmental Mediine (ed. J. B. 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