Mtlbod> rotA>xuuII n••Elf"".0( a..m",,10 oaR"", o<Io« "" F.a<t>o;=Eol,ted b)' v B. V""k.o<I P. J. Sho<ha.C 19lJ SCOPE

The Mamma/ian Embryo and Fetus In vitro

D.A. T. NEW

1 ClJLTL RES \'S TD IS

Cullu", sy' tems have a number of ad"a ntaF for the stud) of mammalianembryO$.They allow dose and conunuous observation at aU ,tages of embry'onicdev.lopment. and of embry'os tOOsmall 10 be obse....-edin the ule",s, They' alsoallow precise control of e~perimental oond ilions. a nd rnablea cleard ini nelion \0

be made bet"~n direct .ff""ts ofa treatmenl on the .mbry 'o .nd tltose r.... ltinllfrom interact ion belween the trealmen t and the maternal metaboli'm

For a culture ,ystem to be of value in a~,ing the effect<of chemicals. it iscssontialthat il be capable of supponing normal p-owth of 'control" .mbryosAlthough .n sta"" of embry'o fetal d",,,lopment can be maintained in culture.there ar eat presenl only I"" periods of de, .. lopmontthat can be ,upported "'tb.high le\"elof reliability. They' are:

(I) egg to blastocyst (preimplantation Slage,j-particularly of mouse andrabbit:

(2) early fetu' (organog<nesi, )-of mouse and rat.

Methods forculturing.us and blastocy~1S ha, .. bttn a'"Rilablefor many yearsand are well known. They provide a .imple method for t.. tinll the elf""'"ofChemicalson the earliest stag.. of d",-.Iopment. Ho".. ' .. r. these preimplantationnnbryos ha'e con'iderable capacity for roco'""y aft.r injury. and it is the lat.rstag.. of d"olopment. particularly during early organogeneo,•. which a re lhemost susceptible \0 permanent damage . Culture method' thai w~l rna,nta,nembl')"OSdurin,l organOllenesi, (New. 1975a) seem lhe",fore likely to be ofpanicular \"alu. for a..... illi embl')'oloxic elfoolSof che""c.l,. and mQ$tof thefollowin,laCCOOntwill be co~med " ith lhem.

1.1 Typn of Embr)'n Cul,a", Slst oms

The main t)l'C' of cultu,.. sy".m, in uSCin our laboratory' .re ,ho"'O ,n fi,lU,..,(a) petridish cultures , The nutrient medium is placed ertiter as drops undor a

l.yor of mintral oil. or is conl"nod in a walChg1." Such oultures with "at.,

277

""'~ .....{ ,]:;:::. £2$.,

7WlI CoIkll1 W

(ion Ball'"

"""......

F;alUl: 1 Em bryO cuJ,urc 1)1­,...... (a) !'Wi <Iil.bcuJ'UrelI. (bl'0"'"!Io.. .. . (e) 1'0\&10<; (d)cir<-uIa'","

O:llco~

-,~= _~'o;;,.••••>.

'"

Goo"")1

Gell Outlet

,,'

Q,'Cj;

(non ·ftowing) medium ,up port Ille growth of ew and blastoc:ysts. and ofpost implantalion embr)'os up to earl)' bmi).bud ' tag.. (25-30 $Omit.. ),

(b ) Roller bon"', The culture ' ..... 1sa re .mall c}'lindrril bonles in whi(hpart of lhe ava ilable volum e i. filled "ith nutrient medium (usu.al1y "n serum)and the remainder with the required O , IN, /CO , mixtur e. The bonJes are laidhonzon tally on ro llers a nd rotaled at 30-60 ",v min during incubation. Thismethod suppo rt ' beu er de ,..lopmen t of pos timplantat ion embryo •. and to moreadvanced ,tages . than ca n be obta ined in ,talic medium. It is alJo part i(ularlyuseful in experi mena to test lhe e1fectof an agent on embryonic development;lhe co nu nuou, moVement co,um maximum and e"COeXpoJureofthe embf) 'o tolhe agenl .

(c) Rota tor. In this ')'Stem .• mall cul1ure bou'" a re attached to a hollo '"ro tat ing drum . The oxygenating ga' ;, pa , sed continu","]y Ihro ugh the drumdurin g the culture period . The method pr<>vid.. a co n$lant 0, a nd CO,ltv c! a ndmInimum "ariation of p H in the cullure m«\ium .

(d) C irculat",..The embryoure a nchored in a smaUcbamber Ihrough ,,'hich aconlinuou, 0 "", ' of medium is maintained . The m<>:IIum Il oxygenaled andcaused to Dow by a meam of bubblC!lpa"':n, up a slopin, tube . Because the

<II; H_<yt;.-, fold

I P~F IUmos and

'.'

••-- - -

8 9 '0 11 12 13 U I S

o~ '" 'lh t O\>O<'l

FiIUl< 2 I'ttiod. of dc>-dopmcnt IDculture of nI,....bryos ' 'Pla nt<d "'dilfertnt . tal" oforpno~,

280 M f/ hoJ. j QFAsu n ing ,n.,EjJu u ojChtmicolson ReprodW:Ii •., F~nClimu

embryos are fixed in posilion. tbe method allo" " lbem 10 he ob5<:r\'edrontmuou~~ throughoullht cultureoenod.

1.2 Du",'ion ond Rote of G","l h

Figure 2 .bows lhe penod. of d"'t lopmenl of po$limplanTalion ...1 embr)'oslhal can he maIntaIned in c ~lture. Embr)OSplaeed In cult ure at "" " ylmdcr (Sday) stage can he maintained for 4-5 days and srow 10 3l}-40 'om ite stagesEmbryos placed in cullure at later stages develop for progres,i.'e ly .horlerperiods,

Figure 3 compa ....... 1.. of protelJl .ynlh ....s by ta,embryos in 0;"0 and in"ir", _ Up 10 the llt..:,lay Slage, tlo: ral. of prol .in '}'nllo:si. ill "ilro i.Indistinguishable from lhal in ,,'vo:~ft.. thIS, the embryos in \""0 grow more5lo""ly (altho ugh ral.. of organ ditlhenllallon remain ....arly normal) . The

.. '.. '" '" H. ".'"II" f,om.""" opt"." Id..... 1

F...~n: J PrO<<ID """I<lil of ral emk)· ... ... planted bet.....,., 9and t4 d.ys of l¢SI.ti"" ond tro " 'D I/O ,j,,,,(0(l<Dcircks ). com paredwilh I' '''''th in n>(>($ohd oircles)

T~ .lJamma/ian Embrya and Fe/us in ;-ilra '"

F,iU'" 4 Top I<fl, bead_fold eml" " ", .. explanl«l al "! days' ~Ijan, TOIlnih t ' Emb.,.", pown for 32 ~oun;. ,i 'Q (upp<r ro" ) and in ,;". (10,,'", row). Allcig/lt <mil!) ',," Or< from the >a m<: ra', Bottom : Embryoo Jro "n for 018boon m';'0(up".' ,o w). nd IJr '; ' rc{Io " . , 1'01',) . All eight embryos . re from 'he" m< rat . (Alltl>< pl>ol OV"p hs . re "' t l>< .. me ""'JI'di<01lon ( x II)) (N, .., " al" 1976b)

282 Mn 1toJJj o. ASHSIIntlilt £jJHnojClwmk"uOft lWprodt..:,;WF"""'iMn

dilfe"""'" IIIJ1O...-tbrate after II! daynl""""t eelU,nly ~,,1~ from the IKk 0'"

fllJ>CoonalaDantoM: placenta in cult"",.

1.3 IWIaIoIIity

The eu1lu",,~ a", ummely "'liable for rat embr)'olI dun.., till: 48 boutperiod bmo='l bead-foldand carty Iimb--bud RaF' (9~-11 1 days) . (}.-er 9~. oftho emb<yoa form a blood arwlat. ::llla..t <k>,,1op lIOrmally duritl& tIbt pmod •.-ith rates of proteUI tyDtlaa a..t dtll"err:ntiatioD iDditwlpasIlal* from tlQe ill..... (New ~l 111_ 19761>). TIIiI ca.a be particularly -a ""-n by companIlI till:<k>elotw..uJI mcultun: of till: cmbr)ooa fromOMuterine bon of a rat .,t.II that oftb< cmbrycJs left to IJOWill tinI in till: other uteril:lo born (F......, 4).

U Sc.iri"I) -

Obsen'atiom on tIM:irnrntd .. te elfeclt of cbcmiab 001eIP a..t blu~.,u IIIcui",,,, can oltea be aUp>eflted by nudy of 10l1li""""" dl"<cu al\er tIM:eg,blastoe)"Su haY<been mllmod to tIM:ut",-,,-

P .. timplantataon m>.bryOJ<;aIIfIot be m"n>ed to tIM:Ilten1a for o:onunucd""'rlopment. Ho~_. atellSl'~ orpDOJC1>CSiaOCCURI', tbJn a 2-3 daywlw..epmod . and by eomlll",nl n>orpl>oJog;ca1and bi'to!olical Ot>servaUOM ..., t.IIdeterminations ofbean rate. protein and DN" content etc .. tbe s)'Stun provMlCla.. nsitive tcs t of the KUon of toxic aFn~, If mc..-e.ucd ..... ,tl\",ly and ordisorimm a tion is n«dcd. it mal' be obtained by:

( I) locreasillj l11enum bers of embryos cultured. Ileca usethe culture '}'Slems arcboth simple anti reliable. it it feas ib le to use 5<1-100 em bryos ror .... h dos.oICof a leSt allem. and rcdetect abnormalities ap pea ring in only a rewpercent ofcxJlO5Cdcmbryoa.

(2) Mak inll deta iled mea.uremenIJ of .. ltcted paru of the cmbl'}'Olll. T hi,method _ • .-J,n a ttudy by Cockroft and New ( 1978) of the efl"cc~ of

Table I M..... p«><ClIl.C1'(>WD- ...... p IcnJIhaDd ......... n"",t>en '" ral tmbryoocaJrw<d a' ddrCftll, , .... pora,_

N." ~ ~

C__"-

T~_ --e_,.. U.% .... ) (..... :1.. . .) Ct .... )

~~ " " II' %10 1.31:!:o.o, 2U :l:1U.. e-c " " 1161: • l.ll%Q.0I 26,.:1 Q.l

""" ", 122 %10 l.6J :!:Q.09 UI tQ.7 '

.I .K ", ~, . 2.\ . :11.06 - '

'. _It.COIIen _'s. __ ,-._~ __ ,l""

Th. J{amma/ian Embryo and F.lIlJ ill ,-ilro 283

Tob!< 2 Frequco;:y of cenain .ba",moli,ics foood in • =bryos cuI'ured ., dilferen',..",pero'o ....

T_ of .baormo~'y

Nou,.. ] fokb -No. of A~_ M icr.,. """; cordi . l ,~ om'T....por:uon: m"'"m.,~ "'~, - oo'_pooI ,.•'.O'C " c c o • •~.O'C "

, , u c c40SC " " to , o •al.l)'(: " " " "

, ,F""" Codroft uol N.... ,m

hypenhoermio. Embryos ""ere ;,.;ubated at 38 C.4O C 40.5 C and 41 e­The .mbr) 'os at 40.5 C and 4 1 C w.r . ohviously abnorm al. Those al 40 Cshowed noel""r malformat ions and the protein con tent was similarto that ofthe controls., 38'C (Ta bles 1 a od 2). How.' ..... it WOJ SUspcc1e<1thatthey ""&hIbe mi",,,,,,,p!lalic, and litis "'... confiTme<lbydeta~e<I co"'Pansonof head measurement> with 'hose of controls, aDd by deteTmi"alion ofheed,<:mbryo pro tein ratios (Figure 5 an d Tables 3 and 4)

0) Injection method .. New and Brent (1972) .xam.iDe<llhe effect of yolk·saca ntibod y (shoeep anti -tat -yolk..... gammaglobu~n) on tat .mb,y"" inculture . At concentrations of 0. 1ma,ml, the antibody causod Sf"'" .....tardation of sro"'lh aDd differentiation. 11"'.,.s suspected that thoeprimary

FilUre 5 Oio.,...,.. obo..;nl <ide 'ie .. 0<1'\) .odfron' .iew (ri.Jht) of b<ad n:Jior>of 1'1' tmbry<><<planted.t ~ d.jo$of ..... tiODar><l""",," '"cultorefor ollI hour>. n., diapam. <bo... m. four b<addimemioM 'ha'"'....n>easUred.od ,he pooiti"" of rhecor mad< for _.t<hea<l .od 1>od-yprotemd.ter min.tiODO, '. hea<l lenll!l. b, lrnJI-hoft.oIen«phaJOD' c. ""-lIhr of .... r>c<pba1oD; d, width.croos te~p/laIo<I (Cockroft ." d N..... 1m)

Table l CompoIi"'" of bu<l 'e","' )'<1J'foteiu <00 10<11'""iooof "'00,," of "" e"'''')'05.,.t eW fortotalJ)l'OIMcontent all.. eulture at ;B and d(l 'C

Tempera .u ...No, of=.,.'"'"

Total emb<)'optotein

l.uI:!:l,e .)

161 ± 9161 ±9

Head,fembr)opt oteUl ra.;o

( :!:•.•. )

O.29S±0.0030.2111±O.OOS

P<O ,02

F'Om C.,. krofl • ..,j N.... . 1971.

etTeet was on the yol~-<ac endoderm Support,nllevide"". " .... ptO\'idod by,njection of the antibod) ' into .. lectod ...g,ons of the COncep'UI. particularlyinlO the amnioti<: """ity a nd extra-embryonic coelom (F igure 10.6). Inneit lw:rof these si' ... ""'1the antibod y in contact with yol~-sac endoderm(although in conlaCt ";tb molt other tM"'" of . he concept"") and lheembryo de'"eloped normal ly,

(4) Prolonged development of pa m of the embryo . In a ",ud)' of the action of S­brom ooro l )'Uric:!II>e (BudR). an a""logu e of the nucleosid e tb)'mid ine.Agnilh and Kocbha r «(976) ul«l a two- Ila ge ""hure procodu .... Whole IIda y mo~ embr)'os (24- 34 somit .. ) " "ere ""ltured for 12-2~ hOUri ina mul ium conlaIlling 50-1SO",g,mJBud R. and the forelim bs ""ere tben .>61«1

FilJU'"6 Inp: hon 0;1.. in nplant> ., 9! 01><1IOj day.¥eI"-tioa , ,0,. ,o,mnioticcavily. B.SpK< betweenomn;"" and )'olk.... (.. t"' ..... b<)'on" _ 10m). He. vy S1ippk, <mb<)'o, Modiumstipp le. allantois. Liaht stipple . o<toplacental cone (N.... aDd8= ,. 19m

T"bIo 4 Coml'll,iMo or head d "''''o>loo ' or l'0""" or "" cmbryo< m.'ched for <rowo romp l<n~'h .ftc""I '" ,,, . '.Ill . rId 40 C

,., .., ,., '"'Lcllll'h or LcOK,h or lleigh' or Wid, h orT~n\p< "' I "~ No, of Crown ru mp ••• 'e I<O';"I'N Ion 'cl<",,<phaloo rcl<occp""Ioo

~.~ (moo:t i,o) (mm :t 0,e,) (mn'±,.., . (m,n ± •.• .) (mm± " • .)

JlI ' I,; " l 19±O.l)( 1.52:t 0,(14 0.6J ± O,02 O,1J ±0 .ll2 LOJ±O.OJ~·C zz J, 19±0.()4 1.42 ± 0.02 O.S7±O,0 1 O,66±O .OI I.OHO .OZ

P <O .OI P <O,02 P < O.OZ

I'm <le<., h or ..... .. " ........" >« F'I U'O'F,orn Cock,.",.od _. 1971

•~.

286 Mn ltodsf o, lls.Hnmg th.>£ff«ts ofC~m~also~ RPp~li'it FWN:tiotls

and cuhu red k pa"ucly for a furtlll'r 9 doys In drull ·frtt modlUm.The ,"uh,sho",~ significant and dose-rela ted inlUbition nf bmb chondrogenesis bythedru Jl.part icular ly in ~mbs take n from embryos lha l had been exposed to till'drull at the 26--29 somite stage.

Further illust~tion of the possibilities of embryo rulture method' for8$$($$inllthe effects of chemica l' will be Il"'en by indicating someof the studiesmade ...·ith tbe5c melhod. in different Iabo~lories. The folJo...inll selection i.inlended only to illu' trat e lhe range of applicalions possible; it i. not acompTChen,i ... re' ·jew. Further d i", u.. ion of both the advantages and limi·tations ofcmb rw cultu re can be found in New. 1975a.b; Wilson. 1978.

2 8l.AST OC YSTS

2. 1 A<tion of Vlrious Agtllt. 01 Ib. bbit B1l1$l<>cys15(Lu t.... k.C\lana .r at. 1 969~

8 1as tocysts "'-eTC ta ken from the uterine horns ofrabbils at6 da y, ofgestat ion.Tile bla,tocysts were transf erred to small wateh glasse:' of culture mod'um In pe" idishes. and inCUbatod in a ps chambet contlln inll 95% 0 , SOl. CO " Eachwatchglau contained 4-5 bla,tOl'Ysts in 1101of medium TC 199 "..ith O.02 mgbo, 'ine plasma albumIn.

Substances tested in this syslem. a1''lIriou s dosag es a nd for ,.. riou, durati on '.1T'O:ludcd ,he folloWIng:

Am imetaboli tes: 2-.de<lxyglUC06e. 2-<Jeoxyglurose+ phos phate 2-<Jeoxygalac­to.. . 6-mercaptopu rine ribos ide, DL..,thionll1e. isoniazid, vitamm BIl

analo gues.Enzyme inhibiton : oL-B!)ceraldehyde. sodium salicylate. bromoacet)'I .o L_

camili ne. jK hloromercuribenzoate . sod ium Ouonde;An'imitolic all"nts : coke mid. aminopleri n ;Cytost atic agents : actinoml"'in D . cytochalasin 8 ;Hormo nes : poly<>eStriol phosphate. gro"'1h hormone .

Results were a=ssed by noting any effects of the l.. t substance on:

(I) expansion of lhe blastocoelic cavity;(2) the embryonic dilC: size and shape, frequency of dividing cells. abnormal

mitotic 6gures. metaphase: arrest . cellu lar degeneration .(3) the trophoblast : frequency of dividin g cells. abnormal mitotic figu," .

metapha .. arrest . ceUular degeneratI on.

Many of Ihe subsla.nces.at coocentration' of O.I -1.0mgJml. showed efle<1HlIlthe blas tocysts an er 6--8hours incubalion. But the pa tlt nl of eWecnvaried fromone mbs tance to another . For e..amp\<'. 6-mercaptopurine riboside caused de_

generallon in II>: embryonic disc bul had lillk dfe<:l on the 'rophoblll$l. 01-­

glyceraldehj<le was more harmfu l 10 trop!>oblasllhan to disc. aDd aminopterindamaged bolh disc and trophoblasl. A few of tlto ail'n ts were damaging alexerp l10nally low eo""enlra'i ons : e.g. p<hloro"",,,, uribenzoate suppn:ssed al lgr<!""' h at I l' l<ml .

Effecls of the ,ub slances in ";lra"''''''limes differed from tOOse in .i.o . Foreumpk 2-<ko~yglucose was lessharmful at lXIuivalent eoncenlf8tions in vim'than in ';'0 . perhaps iDdical;ng that in , ;vo a metabolic modification mayiocrea"" it' act i"" on embryos . C"">er""ly. aminoplerin "as "';lhoUl .tf<ain v;,'o but aI 0.2 milml ,,",ult«! in inh ibilion of <d 1divisIon and degelltralion oflito bla'lOC)"SlSin vi, ,..,.

WIton all"nlS were admi odterod in vi...,a nd the blaslOC)'Otsllton e~ pla n1ed (o nday 6) and cultured in agent -Freemedium. many 0r,lto blastOC)'Stsshowed partialor 100al r=,,'el)' ,

(n-mll. lhese uperiments showed lhal blaSlOC)\ts in cultu", pro. ide a simplemethod for evalu aling toxic d\"ecl' orcltomicals on lhis Sta il' of lk ....lopmc:ntHowe''¢r. ,he effeelS may nOt a lwa)\ be tlto loamel$ i~ v;"o. and if II>: b1aslOC)\lSare nOl too "",-erd y damagt<:! they may reCO''¢r wn.n tn. agent is remo ,"Cd

3 RAT AS O '-IO ll SE D IBR\ 'OS DURI S G O RG AS OG Et'l[S I$

3.1 TribrnnHltfha ..... : Cadmium, Lead, Arsenk

Ka ufman and S'oele (1976) e.a miMd lhe lk' -elopment of hc:ad.fold stage ralembr) 'os exp::>sedin cul1ure 10 the anxslbetic Avertin (uibromoethanol). In •' sta ndard dose' of A' . rt in (0.02 ml of. 1.2%solution of Avmin per ml of,,,,rum)growlh ofille embryos . as asso:ssedby synthesis of ne"" protein. was sipificanll yleu than thaI of controls . Embryos eapoKd to lWice lhe standard do", showedfurt llt r reta rda l i"" of growth and vario"," othc:r abnormalities including poorlyde,'¢loped SOmiles and hear t. and a failure of the nnbryo to rOUl<:10 the fetalposition . The resullS lkmonslral«! a .eralOll"n;c action of this anaesthetic andemphasiHd 111<:clinical importana- o f do:termini'4l wlltlher any of Ihe anaes­.h elic agen ts commonly used in hwnan pra <tice ha, .. similar embryotoaic action....hen admini''''red during earl}' pregnancy .

A sludy ofl lto effects of cadm ium has been made by K"'in t l al. (1980). In """...rieI ofeaperimenlS ""rum " .... prepared from rats al ln", ,",'aloof 1.4.8.16 and 24hours aFler injection of. mi h bul su blethal d"'" (2,13 mg"kg Cd) of cadmmmchloride . Head-Fold .tage ral embryos ....er. tlton i""uba ted in tilt ""ra for 48hou rs. TI>: I hour and 4 hour ", ra ""ere rapi dly ktha llo II>:em bryos. Sera ta kenaf ler 8 hours allowed "''''' inl for 48 !>ours bul lhe nnb ryos .!>owed vario usabnorma lilies. ;ll<lud ing failure ordosurt of the fOK- and midbrain. andretarded growth (f able 5~ In 16 and 24 hoUl"!i""fa.embryos appearedmorphoJosically normal but ma Iler than contro ls.

288 Me/nods/ o, As.Y'",ing,he FJ!fflSo/Chemkol, o~ Rq ,od!J« iu Ftmoiotls

Tab« S Protoin and DNA conl.."l$ of ""bfyoo ",hurt<! fo< 48 hour> on rerumflomIJ.\~ inpel! ""libl.13l1\l!cadm,umti

Hours &fI<f No. rail No . embryos Pro<oin,embryo DNA embryoiqjoc'_ rerum donon .~,... tuc 1 H ) V'I '" •.•,), • • 13.810 .1 0.9 10.1

• , • 12.2 1 U J.S 1 0,3

• , • 68 01 U 6.9102rs , • M.7 1 1.2 6·.10,6

" a • 862 :d1 7,01:0,8Conlrol • • 96.1 :l:6, ~ 1.7:l: 0,6

A<I.ptod 'romKm. " _ .. 19l1O

Thos<reiuh. att 'nl ..... hnll ,n sho'"nll how lilt cuhur. system can be usN 10

d.,.rmi". lhe ehani"" in loxicity of an agent 0'''''a period of lime as il interacts,,'ilb 1M mal.rnal metabolism, Kloin .t 0/. ( 1'180)al$O.... mined 1M elfeell on ralembryos in cuhure of 1.6-2 .0 I' ll. cadmium added 10 ~t\Im from unlrealed "nsTilt .... ponse was qu ile varia ble and embryos in lhe sa"", cullure ,-.ssel includedlhose that wCtt normal. abnormal Or failed to IUr\W. tilt culture period . Tiltabnormal emb')", showed exte""i, .. haemorrhages but lilt neurallubel; elooednormally. Al'ogt thtr . lhe palttm of r-espon~ of'he embryos in ~t\Im wi' h dir<CIaddilion of cad mium was "ery diffe"' nt from lha' in.era from inje<:led an imalll.

Beaudoin arod Fisher (1981) b" 'e combine<!maternal trealment wi'h ;~ ,'U,ocullure in e,,,lualinll a number of leralO$<", includina cadmium. lead anda .... nic. Emb') 'O$".. reexplanleda110 da}'Sof gestal;cm (~9$Om;\e$) from raUlwbich had been lreate<!"'ith lilt agen t 4- 24 houn previo usly. Tilt embryos werethen gr",",'n for 24-42 houn ;n cuhure me<!ium w;lhoul ,eralOilen. In culluremany of thes< embr)"os 'ho"-ed failure or slolO,ngof axial rOlalion aDd neur~1

tube closure. as well as retarde<!lOmite and limb-bud formal ion. compared ,,'itbcultured control embr)"os from unlre ated rats. An advantage of lilt proeedu '" ;1'hat ;1al loW$Iludy of eff""lS of embryonic dnelopmen' follo wing exposure ofthe intacl molher-placenta -embryo uni' 10 an agenl for a brid and preciselydefined penod of gestalicm.

3.2 Oeplc{ion of G__ • "-mino Acid•• Vita mins

Coekroft ( 1979) usoj 'be cuhurt Il .. ,.m '0 lludy JO_ of tht nulne nlrequiremcnll ofra ' emb')'O$ du ring organog~nesis. The ~ulture ~",m ...-asfirstdlalys.<dagai"'" a balanced ... h SOlulion and 'hen supplemenled ..llh "mou Scombinalion. of glucose. P}'","ale. " tamin' and a mino acids. The .... Ulll areshown in Tab l.. 6 and 7.

Gro ..,h ...-a l min", .. 1when lhere was no energy $OUI'OO,but the addilion ofglucose .... ,ored bolh growth and differen'ialion 10controlle, .. I• . The inclusion

T.W.'M _ t o.o.__ """",,, ., __ bcn ... ....-.. _~<J6_It .. ,_,..<al_ro.4I.io._ .. , _ DO"diaI)1o<d...-..........1 "". __ ta. .. _ <J6...... . _ ..... , __ wad_ oads

~. - ~. c............•• 1&_ "" - ...... l_l

4")""..,......... " 15't U ltI 5.03 LllI to..l1l)al l"'«! .. ,wat " I I t l ' 0"... ± 0 03·I.looJl"'«! .. ", .. , I JIu<- u UJt 10 ltI9 tO) HltO ,Oll1)al )'Wll .. ", .. t f Ii .... + I"J"'''''' u 1011± , . 2)lt0 1' 2.61 iO .01·1>. 1)'&td.. ",,,,r +""", .. 'e " :!lI ± S' 0.62 t 0.02 '

(It , WIIolt It ", .. " lnil 2UtO .2 l.ll iO. OlO" I)'Wd...... '" I " 44 ± 4 ' UI :1:0 .17"OIoJyIi<d...", m I + >;10"' ;" ' " 172:1:9 26. 1 t o,' lIS :! 0.081)" lyIi<d...... m: + >;1>"" ... + ' m,no ""oJ, " 13S ± 6 27 1,0,2 lJll±0 04I) ,oll-..d ...... m : + om",o ",, ;d, " SJ ± S' 1.1'9t 0 IS-

.~'''''"''"''y d,n",. , f " .. ~, t"' ""h~~.. "' '''''. !' <:0001t ' '''IIol'''II.....n<>. .. 10. ... "" M"',.' C.. ,... i".' I"I I~_I"1.,.......C"' l' on. 1t1"J, b,l""-... ....low ... of "',.-. ,... ..,j " " ili'l Uti,

T..... l MaI' ± ""-. _""*,,, __ "'_bon..~ ~""II ............ ......a;""III~ .... , ... bryw<u~undr..... ~ III d.1yood .. , -.. ""'b 11_ ._ _ nnous .,...110.,_of .. ,~ ..- - •• .- --•• .- • • ("-- .---- --- - ~, - ~, • - • - -",_" "'- " "'- ')0.0_ ' ) " · .... '1 ~'...,.~,_.."'- ')-. - - ,-~

,Ai"' • • • • • • • • ,""." l 7f ,., H h Ol3• " '" 2.1f • • , J ,

• • • • • • • I ll " ,....... ,• • • • • • • "" " 10"" H',"7• • • • • • ,.,," lU ,01 n. ,o.It, • , • • '" ," "" .. , ,, , 0. 11 •, , • • ,.... 10' •" l "' • • •

• , • • • ,"'. It " ,". H ' " "• • • • • • • • 1'." ,.. , 01 "., 0." •• • • • • • 170 ",.....,.. ,0"

<0,. • • • • • • " ''''' " lOI , OJ • • to Ol

• " ,.,." ll"" 1.>:0. ... ,• • " "' ''" ll ... .l •.".... ,• • • " ' 02 .. 11 llit'l u .....• • • • " , • • II " .. '1 .........-(,,..,,,,,.......... .._., _ __ f_ ,.t.<l

The Mammalian Embry a and Fetus in ';Ira '"of pyruvate with glucose had an .d,,,roe effect on de, ,,lopmont and pyw\'. t,alone <upporte<!"" y hn l, d" ', lopment.

The p"""ntt of free amino acid' appea red to he "'lati'"ly unimportan t. bu'vitamin' ""re essential. The vitamin whose abseoc" produ«<! ,he m", t markedeffect ".-,,, pantothenic acid' protein content and crown-rump length!; we",SIgnificantly reduced .• 00 m", t of the embryos ""'" abnormal. Absence ofriboflavin or folic acid also .ffected gro"' h . nd differentiat ion bUl to a Ie,s«extent, Absence of '-in"'itol caused de>'elopmental abno rmalitIes (open neura llUbe,) but did not reduce protein synthesis,

Cockroft ·, work ha' pro"ided an ':<cellent example of the l'O',ib iht'e'pro ,'ided by th' culture ')~tem for deta iled aoo precise anal)~is of the nutr "'n trequir~menl$ of embryos. It could he ",levan t to any toxicologic. i stud'esLnvol" ng changes in the bala""", of serum cOlIStitutents,

J.J ExC<S!lGlucow: E~C<S!l ~nd Dotid, ," 1"",lin

Cockroft .n d Coppo la (1977. and personal communicat ion) found tha t extra D­ilu= added to tbe cultu", medium in e' ct &Sof 6 m&lml resul"d in severemalforma tions of rat embryos. mcludmg abnormal fu>ion of the neural folds.mi~rocephaly and eye defect>. Tbe glucose le' '' 1sof sucb culture, were similar tothose occurring in the blood of se,,,,,,ly diahetic pat ients. The elf.. ,u seemedspe<:ific to D-glucos<.because L'glucose produ~ no abnormalities of develop­ment in culture and only a general retard ing of growth

Addition of e~,ra insulin to the culwres stimulated embryo growth (20-25~~

increase Lnprote In synthe<JS)wi,hout cau>ing malformat iorul.Sadler (1980) ha' found that S mz,ml extra glucose added to cuh ures of mouse

embryo, <au5ed neural tube defect'. He also obta ined , imilar defects in mOuseembryo' culwred in serum tal: en from ra " rendered d"'heuc "" h ",eptoz01ocin(The serum w. s taken on ly after . Ut race of the drug had disappeared). Theglucose b 'el in thISserum waS4 m",m l but the in,ulin k \'d (9I'U lml) did no tdiff<r ,ignificantly from thai of control serum. In serum from more se"erelydiahetic ra tS(glucose 6 mil.ml. insulin I"U iml) the mouse embryos showed bothneura l tube defects aoo reduced protem synthesis.

Dlab<tes in hu man mothe" is known to result in increa5ed incidence ofcongenital malformat ion' . stlllb,rth!; and abor tions. Until ''''' enlly. a majorob' tacle in the study of the mecllanisms in"ol"ed ltas be<n the lack of anexperimenta l mooel. The ~ul,ure system mak<s pos, ible study of the separateeffects of hyper-glycaemia. hypom sulinaemla. etc . on <mhryo'. and a" Olds anyconfusing effects mulli ng from dru gs (e,g. m eptoz01<><:;n),

Studies by Shepard and his colleagu... at ,he Un"" r>ily of Wa,h ington(Shepard ., ai,. 1970) demonstra ted some of the path"'a~ of energymetabo lism tn the rat embryo in culture. The}'added " C·lI!UC05e to the culturemediumendtracedthe tate ofttnsglucosein termsof the amount of lactate

292 Melho<b10' MM Jsing Ihe EiJeCls ofCnemK:(lls on Reproduct;le Funct ions

'00

'00 E""'yO·""'"~00 ,

, Laot" ' .

I,e- •t ' 00I, zoo

' 00

"0"F'iur< 7 Utilization of i1"""'" byrat embryOi in cuk.", (Roproduocdfrom Sh<pord" ai , 1970. by pcnrtiMion0( Ae.... mic P..... I"". )

released imo the medium. amou nt of respir6tOr}'carbo n dio' iM iI' " off. and theamoUnt thai finished up in the emb,yo and membranes . Tn. .m bryc» were ofthr« d'fferent ages. 10.11and 12day" "..hich approx;mately' ' pans the p""od ofmajo r organogene si, . The re'uh , (Figur. 7) showed that durtn g thiS r><riod.rale, of g1u<X>se upt ake and lactate product ion are dcdin ing rapidly "hlCh.together "..ith other "' tMoce, Strongly suggests that lhe energl pathway, areohanglng over from anaerobic glycol>~i , to the Krebs O)1Z!e and olectrontran, po rt .>~tem ,

In a study b) Robkin and Cockroft (197 8) , emb') 'OSwere cultured for 18 hou"in a gas mixtu re <X>ntainlng 1-10' .; car bon monoxide Althou gh differentiation(",m ito forma tion) wa' unaffected, the gro" th rat< (pro te;n ,y'nthesis) wasreduced, ""hile glucose consumpt ion. Iacta to produet ion and the lactate glucoseratio ""ere all increased. S"n, lar but more pronounced effects ",'ere obta ined inculture. where theCO "-asomitted and the 0 , of the gas pha.. reduced be low theop" mum 1<>'cl. The two sets of results indicated that under anoxia. resulting fromeither CO poiw oing or from reducN en"ironmenta l 0, . the midterm embr yocan adjUStit$ metabolism to obtain more energy from anaerobic gll1Zoly$ls tocompen$.1tefor the reduoed aotivit)' of the Kreb', cycle and electron transp ort$Y'tcm. The compcn$.1tioo i' only p"rti al "od IS,nsufficient to support normal

TM JfammalitJn £rob,}o and &rus in "lIFo

ral'" of protem .ymh""i,. bUImay someumes be adcqual<lo ' Upporl differentia­lion of organs similar 10 'hal ,n con"ol embryos,

3.4 h~Osn'"

At thc carhcr "ag'" of organOjl<"n",is.rat cmbf)'os dO\"lop normally in cullure ina 11'"pha", conta,n ing S-10". o~}gen, This is cqui,,,l<n, to o~Y'gen pr",su"",,be,,,ttn aboul 40mmHg and 8OmmHg. "hich are dose to II>:,,,lu,,, usuallyfound In 'he ulennc ,,,,n and arlcry . r",peeli"c ly. When 'hc o~ygen is ra'S¢<!to20"0many of'he embryos '00" a failure of closure of 'hee'l'ha lic neural fold, . a,in lhe carly stages ofe,~ncephaly (Ne" el al.. Io76a). Thi, suU"" tha t e~posurcto hul< more ,han t"'ice ,he normal amoun' of o~}'gen issufficient10cau.. majorabnormahtn of dC\..lopm~n,. The effeclson lhe neual folds .rC .,sociated wilh1055of apICal mICrofila.... nl bundle!; from the Iat.r~1 «\Is. and reduction ofneural c,,",,' mlgrat,on and p<Oj!rammed~\I dealh (\torn" and Ne". 19791,

3.5 E:rr.,,1Sof T~mptralurr and Drugs on tho Hr.rt R. lr

Detailed records of ItIchearl beat of the early embryo "ere obtai ned by Rob!:,n01ai, (1972). Ra' Cmbr)05 e~planted at 1\ Jda)'1 of ge"at'on ,,"r~ euhurt<! ,n ac,rculat,on ,}.t~m and lranstllumina ted wilh a 1o"-p<J"er Ia",r beam, A lensfocused the image of lhe embryo imo a pbot omultlpl)ltli tube and lhe nic!:"­prod_d by II>:beau ng hc." could then be OcICClc<lby an O$CIlloscopeandrecorded 011a strip charI recorder (Fiiure 8) Heart rales (i.c. lhe fr"'lucn"",of heart beat) m = bl)05 of this age at 38 C" ere aboul 160per min bUI wilh'0 .... '''rialion from one embryo 10 another: lo ".-~ring the tempera ture ga"~ areduetion in heart rale of .bout 7". for each degree drop in lemperalure

Rob~in er al. (l97 4. \976) also studied tbc respan .. of 'he =br)onichea" to dru g, which are known 10 affect ,he hean ra'e of ,he adult. Tbc drugs" cre added to thc circulating medium of <uhu"," of 10; . I I day a t embry 05(F,gure 8), The addlt,on of ,soprolcrenol. a droll which Stlmulales lheadrenergiC bela-receplors. """hed In an immediate increa'" of heart rale in 10lday embry"" (lO-IS ..,mites), Th~ effect could be quickly lerminated by lheadministra tion of propra nolol. "hich <ompoliti,'ely blocks lhe adrenergi< hela­receptors. bul "hich did not prnenl • further increa.. obtained b) admlnislerln gtheoph)·ninc . )'le tho 'amine had no effect,Trials ,,'ilh atropine. carbamylchohne.Curare (d·tul>ocurarinc chloride) and nico'inc dcmonstraled lhe development ofcholinergic receptors in the heart about half a day laler (11 da)~. 21 2650mites)'han tl>: formation of thc adrenergic receptors,

The'" studies by Robkin ha"e indicated lhe r~asib,lity of u,inll the cultureS)'1t~m '0 detecl elf« .. of to~ic cl>:micals on cardiac pb~~iology from lhe ~arhest

slages of he.,.1 fOrmalion in lhe ~mbryo.

."

•......

•

,, • PI'

.-.''.......

, , ,:- ...~-_......

' .........-.......,~" ,...

II ,~

i __• .- .M.

i ec

i

F" C.I Stnp cban~d looan.-' olll -<1&l "' -"' '' '''''_ (b )Ibn ,_ to<WJ<rml..... (k<prodlo«d &<. R__ ~ • . In ,"'- bypa • <IiMoooL J.-. 1l1li:." "'" s.nn,,..4. 'J~_ V_ I

.3.6 C,'N.'"plP'"h 15 ....,uCSl.bhbcd thai adm>n'stu!1OfI of qdophosphllmodc 10 preJDanllaboratory rod.., ,, cau.c. embryo malformations. bUl (ben: has been cOll­1rO''''~Ya,10.. bethel"Ihec)<'lop~phanudc affect' the embryosdll'cctl) or onlyafter :0<:1"" 1100 by the malerna l metaboh'm.

F. ntel cl al. (1979) !Ia,..,usedrat cmbrycs In cuhure 10~I,.., lh IS. Embryos"'ere uplan ted al head ·fold , lage and ll,own in human .. rum ,n a .o ller·bo nle'j'SlC'm. T" [lie cult ure; ""ere added ~ariou. combina tions of cyclophospha mide(CPl, h .... m;cro scomc fri>Clion (5-9 f'''''lion). and Co{JIoCtON for a moooo~y'

llt'naw .ys tem (NADP H and JllICO$Mi'i'hoophale)Table 8 oumm.anusthe '($\Ill•. No malfonn.ations or .i",mcanl dllf~

,n Jl"M'U nlte '"'en: fOllnd ,n cmbr}os 1JI sel'\Im onl) . sen.un"ithS<9_ roI'aetOr.i. serum 1II'lb ~dopbosplwni<k alone . 01' sen.un ..-;th

TilL Mammali(1n Embryo and F.,U3 in ,'11M

Table 8 En''''''' of vaoou' e<>mbin.lIon,of <ydopho.phamide,and cofactor>on embryo ~oW1h in <ultu,"

C)'dophosphamide

"CofaOlo..Embryo prolcin (<IllMalformation. r/. )

_pled fro m 1'0." 1" .,,, 197\1

on012

•'"'0112

'"S·9 li ' . r froeti<>n.

• •• ••~ rzs0/18 12116

CP _ S-9. Only " 'hen CP$ -9 and ""faClors were all pre",nt , "'ere the cmbryosaffected , The number of malfonnat;on< and amoun t of growth ...tardat;on;ncreased ",-ilh inc...a.ing con<:entral;ons of the CP

The experimen t demOnltrale<l that c)" lophosphamode requires act ivationhefo... ;t becomes le"'togen;c, probab ly by a cytochrome P-450 dependen tmonooxygenase <",tern present in the li"cr.

3.7 Deteefion of Toxic Chemicals i. Human Serum

Human .. rum ha$ gennally been found to be a poor medium ror cuhuring ratembryos at the earliest stages of organoge nesis. How.. "r, Chalot" al. ( 1980)ha ,-efound that the embryos develop satISfactori ly if the ",rum is supplemcntedwith add ;hOnal glucose (2-3mg ml).

This has enab led these worke.. to use the culture <",tern for detectingteratogen, or Olber embryOlOx;;,agnn< in human ",rum. They ha,-e 'h""'n tba t

Table9 Growth of ral =bryos in stNm from ~ti<nts rc«i"ini cancer ohemo­lherapy or antieonvulsanlS

No. No. ProleinSubject embrl"" .bnorm al (Jlilembryo)

Conlrol, ," us " "C.DCer ehelDOlherapy

Vincrioline,ek:. ", lettlal •(posl-t.... uncul) " , o "'(no trealmenl) " , o "MOIholr<X"e,etc. " ; ; ~

Tamo.if. n " ; , ssAnt;oonYul<anu

TeVOIol ,. ; ; saP"jOnon. <te. " ; ; "Primidooe... . " s a "Teve"' l etC . " , ; asT·I",,,, lete. " , , n

Adanl«lfromCIootol".1..""

296 M~'hod.l f or A,,, ,,i>lg ,IJ£EJJ~clJ ofClJ£mkals o~ ReproduClIW'F"'''''it:ms

>era fro m patient' undergoing cancer chemotherap y. and pat .. nts receivinganticonvulsants are olien kIM! or teratogenic to embryos (T. b1e 9). Thework has pro,ided a "a luable method for studying the embryotoxic ;ty of t heproducts of interact ion between. chemica l ' ubst' ""e ~nd the human meta­boli,m , But one problem to be o, 'ercome " the gruter ,"~riab;lity of embryogro"th in 'control" human ""rum (panic ular ly bel"-een sera from differentind"idua ls) than in rat =um

AJ'I"h . N. D, a"d K<>e h~ar . D. M. (l976) . D<r«\ "'P"O"re of P"Otlmpla"lat i"" moll«embryosto S-bromodoro,yuridi", in . il r o .nd it>eff"" on , ubsoequon' chondrolen"";n ,he limbs. J, Embryol, e:<p. Morpho!, .16, 623- 6.\8.

Beaudom. A. R. and Fisber . D. L, (l9 SI) . A" '" ',-ro,i" -,;,roe>'aluat,M of leral0 a<niooo<l;on. T"a'oioF 23. 57-61.

C~atot.c. L.. Kle;o. N W., Pia,, ~,J , ond Pierro. L. 1 (1980). S"""", . f"J Oul1ure ofralembr;os on human serum' Use in ,he del<CllO" of 1"-'10110"' . Sci" ta ( Wo</li>lgr<m,DC)N7.147 1 1413

C<>ekroft. D . L. ( 19~). NUlrient >'eqweme nt.<of ra,.-mbryoo "" <leri oioi o'i'lnoK<ne."'" ,;lro.J. flep rod. rmii . 57, 5O~~IO.

C<>ehofi. D. L and Coppola . P. T. (t977) . Tera loaen", effoet.<of ""res< i1ucooe0" head-fold ral embryo< in cullure . TeralOioJy 1~, 14 1-146

Cockro ft. D L.and N", .D. A. T. (t 978). Abr><mNli' ie' indu<e<li" outtu,ed TOtembr~'O$by hypertbermia. T"," oloty 17. 277- 2&4

~ an"'t, A. 0 .. G<e<no",'oy. J. C, lucia .. M. R. ond Shepard. T. H. ( t9 19). Teralog",",!:Jj<>acti.. t1onof cyclophoopho ml<!eill 'il<o . U!~ S# ~, ~7_n.

Kaufma n. M . H, and St ocl<. C. E. (191~), Ddel<fiou. effect of an an, .. ,bet Lcon cuiture<tmommoti," e-mbr)oo NOJ.,.. (La ."",) 160, Jijl _784,

Klei" . N. W., Vosler. M A.•Ch ot"', C L. o"d Pierro, L. J. (1980). Tbe useof cullured r'"embryos to ....alu. te lh , ""0101"""' act,,';,y of .. rum : codml"m and oy<lopllos­ph.m iJe r eraloiolJ 21, 199-208.

Lu'''' .... -M'a" ", C .. Hoy, M . f. ond New, D. A. T ( 1969), ""',ioo of various a~"" 00"'bbl' bl.... oc~~to bt ,i ,c and in .i'TfJ J. lIPprod. r ' '' ii. t8, 2J~-23J

Mo' ..... . G . M. 000 N", '. D. A. T. (t969) . Elf"" of O'~'ie" oo.-.ce",ro!ioo OIl""''''bo­iet'".is of eran,, 1neu",1 folds and "" u",l em l in cu!tured ra t emb, yo>.J. Emb';-olapM crp/[o; . 54. 17- 35.

New. D. A T (1978.) , Whok -embryo cul,u", ond 'b< >lud y of mammalion embry osdU" "i orgonogeneoi •. (1;,,1. R.. , Ca"';'rld~ N#, -S.x. 53. 81- 122,

Ncw. D, T, A . ( 1978b). WOOl<<mbl}'<>explants aOOtran.pla nts. In Wilson, J. G. • ndF"""r. F, C. (Ed. ,) Hafldbook of TeralOlogy. Vol. 4. Chap"" 4. Pl<num Pres" NewYor~ ,

~ew. D. A. T . and 8" 01,R. L. (t 972). ElI'ec' ofyol x-<-oco. tiOOdy0" ra' emb,.,.os 5"'''''"I" oultu", . J . Emb,;-ol. ap. Mo,plIoI. 27, 543-553.

N", ', D . A. T.. Coppola . 1'. T.• 1IdCoctro/!, D. L . (l 9760) lmpro"'" develop"", n' ofhead fokl '01 <mbryoo in culture m ul' ini from low o'Yl"n.nd modLli<;otlollS of thecullU" "",m . J. fI "prod. rm;l. 48. 219-222 .

New, D A. T.. Coppo la. P. T, . nd Coc krofi. D. L ( 1976b). Compo'ioon ofl"'wtb ill,,'''' .nd '" . , '" of jX>S1-irnplo"uho" ' al embl} ·oo. J. £"';'ryd. ap, J/arphol . 36,133-144 .

m

,~-

R....k,... M A . ...., Cocl l'<llt, 0 1.-( 1971), Tho011""", <JfcarboD1DO...w.k <1'11"'­...... 1>0_ &DelI"'W!hof ........ br)<Jo.T~ .t, llJ-).tl

Robk.... M A Sbq:cd .T H _ Bau.... O \191.) A_"'drua.lf«UllIl'holanIll.. of early ... .... br)'oo.T~ t . lS--4l

Robll..... lot It. . Sb<pot<l. T. Ii . ... O)'tt , 0 C(1976k Au_"r=pl<IRdtho .. rty""embryo bean - .,..,..u "", _~ tw<. s...-,.;y.1JoDI.M~ I! I. m-101.

RoM M A .. Sbepafd. T. tL andT...- ... T ( Im)-A _ -. _<UIrJon, ""'hDoq...for embryoo. T~ So:l61-JlI' .

Wlor . T. W (19IOk El"ocu <Jf _lOnoaI doobelco... <ally _,.,.,..... "- pot_ill ' of daabtcl< ........ II H,P" "" • -.I"""" ..T_"*o ZI. H9..J.4J ...., l*-H6

SIo<pord. T H._T.- .... T ... 01RtIbll .. . M A . (191Ok EDnr-"""•• r ill tatl)_ ... aIIIllr)ooI..~L '* s-I •. C 51.

Wi_ . J G ( 911)- Sotnocrof.lo _ .,..... IlIn- pooco.-l _ ...... ". ' y'">='''I I• .....--. J.G. _ F~ Fe. lEa l " • · "'T~. \ '0/. . ,Qaptu!. PImwII r- . Ncoo' YOIt