Blood Imp 3

8/3/2019 Blood Imp 3

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T H E H I S T O L O G I C A L D I S T R I B U T I O N O F T H E B L O O D G R O U PS U B S T A N C E S I N M A N A S D I S C L O S E D B Y

I M M U N O F L U O R E S C E N C EII I. TH E A, B, AND H ANTIGENS IN EMBRYOS AND FETUSES

r~ o~ 18 M~ IN LENGTH*,BY ARO N E. SZULMAN, M,B.

(Prom the Departm ents of Pathology, Harvar d Medical School, and theBoston Lyin g-In Hospital, Boston)P ~ T z s 5 9 TO 62

(Received for publication, Oc tober 24, 1963)The gr oup an t igens A, B , and H in the t i s sues and sec r e t ions of adul t s and

la r ge r fe tuses have been desc r ibed in two pr ev ious commun ica t ions ( I , 2 ) .Relatively tittle is known concerning the blood group antigens in early stages ofintraute rine life. The inform ation available in th e literature concerns the A,B antigens

of the erythrocytes, demonstrable in fetuses from the end of the 2nd month (3, 4),from 7 to 50 cm crown to heel (5), and during the period of 3 to 6 months (6), whilethe w ater-extractable antigens of the tissues and meconium were reported for fetusesfrom 5 to 9 m onth s (7), 3.1 to 44 cm crown to heel (8), and in tissues of fetuses fromthe first half of the 3rd month of pregnancy onward (9). Mo re recently, ceils fromvariou s organs of fetuses above 3 mon ths of age grown in tissue culture w ere shown topossess the A, B antigens by the mixed agglutination technique (I0).

T h e p r e s e n t c o m m u n i c a t i o n is c o n ce r ne d w i t h t h e m a p p i n g o u t o f t h e A B Hant igens in the t i s sues and wi th the e luc ida t ion of the i r behavior dur ing theea r l ie r s tages of in t r au te r ine l i f e , cha r ac te r ized by the ea r ly deve lopment ofsever a l o r gans f r om the i r p r imor d ia and by the matur a t ion and as sumpt ion off unc t ion by many o the r s . By desc r ib ing the na tur a l deve lopmenta l h i s to r y ofth i s s e t o f we l l def ined gene t ica l t r a i t s , which a r e now becoming t r ans la tab lein to chemica l t e r ms ( 11- 13) , i t hopes to cont r ibu te to the under s tanding ofgene act ion on cel l and t issue level and to the elucidat ion of i ts role in emb ryonicdeve lopment . I n a mor e pa r t i cu la r s ense , impl ic i t in the na tur e of the an t igensunder s tudy an d the i r homologous an tibodies , i t under l ines the pr oblem of the i respecial biological s ignif icance for fe ta l-mat ernal re lat ionships .

* Read in par t at the 4 7th Annu al Meeting of the American Society for ExperimentalPathology, Fed. Pro., 1962, 21, 69, and a t the 9 th Congress of the International Society ofBlood Tmasfusion, M exico City, September, 1962.This investigation was supported by R esearch Grant A I-3554 from the N ational Insti-tutes o f Health, United States Public Health Service.

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Published March 31, 1964


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5 0 4 A~ B~ AND H ANTIGENS IN IiUM.AN EM[BRYOSM a t e r i a l s a n d M e t h o d s

Embryos and Fetuses.--Embryos and fe tuses from 18 to 125 ram. C R (crown to r um plength) encom passing the span from 5 to 14~-~ weeks O A (o vulat ion age) x were avai lab le a tthe Boston Ly lng-in Hospi ta l . T he lower l imi ts of the series represent the sm al lest (18 mm .)and the youngest (5 weeks) specimens avai lab le . The upper l imi t was chosen arb i t rari ly , asrep re sen ti n g t h e s t ag e a t w h i ch t h e ad u l t s t a t u s w i t h re sp ec t t o t h e A B H an t i g en s i s v i r t u a l l yreached.

A b o u t t w o - t h i rd s o f t h e sp ec imen s w ere p ro d u c t s o f t h e rap eu t i c h y s t e ro t o mi es o r o f l ap -a ro t o mi es fo r t u b a l p reg nan c i e s; t h ey w ere o b t a in ed , b l ed o u t , an d f ro zen w i t h i n an h o u r o fsurgery . Acceptable specimens from spontaneous abort ions were processed wi th minimumdelay .Freezing, Cutting, and Staining of Sextions.--Rapid freezing was effected in a rout ine man ner,as described before (1). Whole embryos or b locks prepared from larger specimens were cutseriatim a t 4 mi c ra i n a c ry o s t a t . To p o g rap h i ca l o r i en t a ti o n w as ach i ev ed b y m ean s o f sec ti o n sf ro m se l ec t ed lev e l s, q u i ck l y s ta i n ed w i t h G i emsa s t a i n , an d k ep t fo r p e rman en t co l l ec t i o n .

Sect ions were f ixed in acetone as a rout ine , but a n a l tern at ive quick d ip in formal in (10per cent ) , fo l lowed by a was h in buffered sa line, was often found n ecessary when deal ing wi tht i ssues hold ing mucus. Sta in ing was effected by using a s ingle or a double layer method.Autisera and Conjugatcs.--The p r i n c i p a l reag en t s , an t i -A , an t i -B , an d an t i -H co n j u g a t e swere as described in d eta i l p reviously (1 , 2).

Severa l addi t ional sera were used in conjuct ion wi th the principal reagents . A rabbit anti-Aserum, p rep a red b y t h e me t h o d o f G l y n n e2 aL (1 4 ) h ad a t i t e r o f 2 00 0 ; emp l o y ed w i t h a g o a tant i -rab bi t con jugate as a s ta in ing layer, i t gave a m ost powerfu l speci fic f luorescence. A fewhuman hyperimmuue anti-A and anti-B sera w i t h a m i n i ma l f i te r o f 1 0 00 b ecame av a i lab l e d u r -i n g t h i s s t u d y t h ro u g h t h e co u r t e sy o f t h e B l o o d G ro u p i n g Lab o ra t o ry o f Bo s t o n an d w ereused on random sect ions from al l group A, B specimens, wi th a horse an t ihuman g lobul inconjugate as a s ta in ing layer. These addi t ional experiments were included in order to confi rmfurther the resu l t s obta ined wi th the principal reagents; no devia t ion from findings obta inedw i t h t h e l a t t e r w as ev e r en co u n t e red .T h e goat anIi-rabbit globulin conjugate w as p rep a red u s i n g a se ru m (o b t a i n ed t h ro u g h t h ek i n d n ess o f D r . S . Lesk o w i tz o f t h e M assach u se t t s G en e ra l H o sp it a l ) b y co u p l in g i t w i t hfluorescein i so th iocyanate . The horse antihuman globulin conjugate coupled with fluoresceini so t h i o cy an a te w as o b t a i n ed co mmerc i a l l y (Sy l v an a Ch emi ca l Co mp an y , O ran g e , N ew Je rsey ) .I t e l ic i ted no s igni ficant backgroun d s ta in ing in embryonic and fe ta l materia l , no doubt onaccount of the low concent ra t ion of g lobul ins prevai l ing therein .Al l sera and conjugates were absorbed wi th hum an o r an im al l iver powder, repeate dly i fnecessary.Control Sera and Conjugates.--All t h e an t i -A BH reag en t s w e re u sed i n co n j u n c t i o n w i t ht h e i r co n t ro l co u n t e rp a r t s p rep a red b y ab so rp t i o n w i t h h o mo l o g o u s e ry t h ro cy t e s o r sec re t o rsal iva (1).Grouping of Embryos and F etuses.--Human h y p e r i mmu n e an t i -A ,B an d Bo mb ay se ra w e reemp l o y ed fo r ag g l u ti n a t i o n o f emb ry o n a l o r fe t a l e ry t h ro cy t e s . Sa t i s fac t o ry re su l t s w e re co n -s i s t en t ly o b t a i n ed , p ro v id ed t h a t t h e ce l l s w e re w ash ed t h o ro u g h l y an d t h a t t y p i n g w as p e r -fo rmed o n t h e same d ay .

RESULTST h e A B H a n t i g e n s d is c lo s e a r e m a r k a b l y r i c h p a t t e r n , d e m o n s t r a b l e i n t h e

e a r l i e s t g r o u p o f s p e c im e n s , 1 8 t o 3 0 r a m , 5 t o 7 ~ w e e k s O A ( T a b l e I ) . T h e1 O v u l a t i o n ag e i s u sed h en ce fo r t h a s m o re t ru l y rep re sen t i n g t h e ac t u a l ag e o f t h e ' co n -ceptns. I t i s ca lcula ted as tak ing p lace 2 weeks after the 1st day of the last menst rual period .



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ARON E. SZU L~N 505group is charac ter ized by the widespread dist r ibut ion of the an t i gens ou t l i n i ngthe cell walls of the endothel ium and of sundry epi the lia , whi le the water-solubleant igens appear a t the stage of 35 to 40 ram, 8 weeks OA.

T h e endothel ia l ce l l wal l ant igens t h roughou t t he ca rd iovascu l a r sys t em a repermanent and persis t in to postnata l l i fe . The epi thel ia l ce l l wal l ant igens ofthe ear l iest group of specimens are found in the epi the l ia of the integument ,the pharynx, the a l imentary, respi ra tory and lower ur inary t rac ts , and ofthe mesonephric and Mii ller ian ducts . Th ey present a pa t ter n o f maximalant igen dist r ibut ion since cont inuat ion of these ant igens into adul t l i fe i sseen only in the epi the l ia forming st ra t i fied squam ous, t ransi tional , or s impleconfining membranes; i .e. , those of the skin , the pharynx, the mouth and theesophagus, and of the lower ur inary t rac t f rom the col lec t ing tubules to theure thra . On the cont rary, the ce l l wal l ant igens of the epi the l ia of the gast ro-intest ina l t rac t , inc luding the pancreas, of the thyroid and pi tui ta ry (anter iorlobe) , of the respi ra tory and upper female geni ta l t rac ts undergo a gradualrecession upon fu r ther developm ent of the fe tus. Chronologica lly , the w aningof the antigens can be said to coincide generally with the morphologic differ-ent ia t ion and funct ional matura t ion of the organ concerned, as se t out in asystemat ic presenta t ion below.

T h e water-soluble ant igens appe ar re la t ive ly la te , a t the 33 to 40 mm CRstage , borne in the ear l iest secre t ions of the developing sa l ivary glands and ofthe gast rointest ina l t rac t . The ant igens of the pancrea t ic secre t ions are thelast to appear , becoming demonst rable a t 100 to 125 mm CR length (13 to 15weeks OA) . Two ca ses o f aberran t secret ion (15) in sa l ivary glands (submaxi l -la ry and subl ingual ) were encountered (Table I ) , wi thout the correspondingphenomenon being observed in the secre t ions of the gast rointest ina l t rac t .

Bo th t he H and t he A,B an t igens confo rm to an i den t i ca l spec t rum ofpat tern s de term ined by the developm enta l s tage of the individual . The re la tionsof the H to the A,B ant igens have been invest iga ted as descr ibed in de ta i l inthe previous study (2) , by comparing the intensi ty of f luorescence obta ined innon-O group t issues wi th the B om bay and the ant i -A,B conjugates, respec-t ive ly . The consistent para l le l of the H and A,B ant igens demonst ra ted there inis confi rmed for the present ser ies; the epi the l ia of the anter ior lobe of thepi tui tary gland and of the lung present specia l quant i ta t ive fea tures.

I t i s convenient to present the de ta i led resul t s separa te ly for (A) the ce l lwal l ant igens, as found in the youngest group of embryos, 18 to 30 ram, andthence t rac ing the i r subsequent behavior , and for (B) the water-soluble ant i -gens, s ta r t ing a t 35 to 40 mm CR, the stage of the i r f i rs t appearance , andsimi lar ly fol lowing them throughout the ser ies .A . C e ll W a l l A n t i g e n s

T h e E n d o t h e l i al C e l l W a l l A n t i g e n s . - - T h e s e ant igens ful ly out l ine the cardio-vascu l a r sys t em by ready s t a in ing o f t he endo the l i um in t he hea r t and i n



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50 8 A, B, AND H ANTIGENS IN HUMAN EMBRYOSvesse ls of a l l ca l ibers (Figs . 1 to 3) . The r ich capi l la ry ne twork in the adrenal(F ig . 24 ) a t t e s t s to tha t g l and ' s a l ready e s t ab l i s hed endoc r ine ~nc t ion inc o n t r a s t t o t h e c o n t e m p o r a r y , m o r p h o l o g i c a ll y p r i m i t iv e , a n d r e l a t iv e l yavas cu la r an te r io r lobe o f the p i tu i t a ry (F igs . 7 and 8 ) o r to the thy ro id (F ig .11 ) . T he l ive r s inus o ids , (F ig . 3 ) l i ke thos e o f the adu l t o rgan , t end to s t a inw e a k l y , p r e s u m a b l y d u e t o r a p i d p o s t m o r t e m d e g r a d a t i o n o f t h e a n t i ge n s .

The Ep i the l ia l Ce l l Wa l l An t ige ns . - -The se a n t i g e n s a r e a t t h e i r m a x i m a ld i s t r ibu t ion in the ea r l i e s t ava i l ab le s pec imens , 18 to 30 in to , and a re be s tde s c r ibed a s s een in tha t m orpho log ica l ly v i r tua l ly homogeneou s g roup . T heyou t l ine the va s t ma jo r i ty o f ep i the l i a , t he ch ie f excep t ions be ing the a l readymorpho log ica l ly ma tu re l ive r (F igs . 3 and 4 ) an d ad re na l (F ig . 24 ) . T he ce ll so f the cen t ra l (F ig . 10 ) and pe r iphe ra l ne rvous s y s t em a re a l s o f ree o f an t igens .T h e hemopoietic elements, b e s t s e e n in t h e l i v e r a n d t h o u g h t t o b e o v e r w h e l m -ing ly e ry th rocy t i c (16 ) , s eem to pos s e s s l e s s an t igen than the ep i the l i a l c e l l sa n d a r e b e s t d e m o n s t r a t e d b y s t a i n i n g b y a d o u b l e l a y e r m e t h o d ( F i g . 4 ) .The epidermis of the integument (Fig . 5) shows c lear specif ic s ta ining whichcon t inues in to adu l t l i f e . T he ce l l s o f the mul t ip ly ing l aye rs rema in fo r s omet ime morpho log ica l ly und i f fe ren t i a t ed , and the abs ence o f an t igens f rom th ebas a l l aye r , a s s een in a l l s t r a t i f i ed s quamous ep i the l i a , does no t become ap -p a r e n t u n t i l t h e 4 0 m m s t a g e ( F ig . 6 ) . The buccal epithelium re s emble s tha t o ft h e i n t e g u m e n t . T h e e p i t h e l i u m o f t h e nasal cavities i s a lso la rgely derivedf r o m t h a t o f t h e i n t e g u m e n t a n d r e t a i n s t h e a n t i g e n s u n t i l t h e t i m e o f a p -pea rance o f muco id gob le t c e l l s , be tween 50 to 60 nun CR. T he ep i the l ium o fth e internal ear, de r ived f rom the ec tode rm by a p roces s o f s u r face invag ina -l ion , demons t ra t e s the ce l l wa l l an t igens o f t en in an i r regu la r f a s h ion ; theya r e n o t t o b e f o u n d a f t e r t h e 5 0 m m C R s t ag e .

T h e phary nx and the rema ins o f the pouches a re l ined by a th in , gene ra l lytwo ce l l deep ep i the l ium r i ch in th e ce l l -ou t lin ing an t igens wh ich a re re t a inedi n s e c t o r s d e s t i n e d t o b e c o m e p e r m a n e n t l y s t r a t i f i e d o r " t r a n s i t i o n a l . " T h eEus tac h ian tube a n d t h e tympanic cavity de r ived f rom the f i r s t pouch a re l inedby a th in ep i the l ium iden t i ca l w i th tha t o f the pa ren t v i s cus . T he ea r l i e s tp r i m o r d i u m o f p h a r y n g e a l o r ig i n , t h a t o f t h e thyroid, i s a t t he ea r l i e s t s t age o fth i s s e r i e s a s o l id ep i the l i a l c re s cen t w i thou t a c ina r fo rma t ion and wi thou tthe cha rac te r i s ti c r ich cap i l l a ry ne twork o f an endoc r ine o rgan . T h e pa ren chym aread i ly demons t ra t e s th e ep i the l ia l ce ll wa l l an t igens (F ig . 11 ) wh ich wane a tthe s t age o f 70 to 80 m m C R (12 th week OA) ; a f t e r th i s t ime , the g land s howsde f in i t e a c in i and beg ins to re s emble the f ina l adu l t h i s to log ica l p i c tu re (F ig .12). The thymus (F ig . 11 ), an exc lus ive ly ep i the l ia l o rgan a t t he p re s en t e a r lys t age , i s r i ch in the an t igens wh ich pe rs i s t pe rmanen t ly in the Has s a l l ' s co r -pus c les , i n con fo rm i ty w i th the re s t o f the s t ra t i f i ed s quamo us ep i the l i a e l se -whe re . The parathyroid glands, of ten d i f f i cu l t t o iden t i fy w i th ce r t a in ty , s eemto poss es s the an t igens ; t he s e , howeve r , a re no t de s t ined to becom e pe rm-anen t , a s in g l ands f rom s eve ral adu l t s no an t igens cou ld be dem ons t ra t ed .



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ARON v.. SZU L~N 509The pi tui tary gland: The anter ior lobe c lear ly be t ra ys i t s or igin f rom the roof

of the stomatodeum (Rathke 's pocket ) ; the ant igens out l ine the ce l l s of thesol id cords of the developing organ (Fig. 7) unt i l they commence to recedeabou t t he 60 mm CR s t age , 10 th week OA. From 12 weeks onwards t he pa r -enchym al ce ll s a re com ple te ly f ree of the ant igens (Fig. 10) . Through out theposi t ive interval , the A,B ant igens are consistent ly markedly weaker than theH ant igen (Fig. 8) . The poster ior lobe , der ived from the bra in , i s f ree of theantigens (Fig. 9).The esophagus (Fig. 11) i s l ined by an epi the l ium which in some of theear l iest specimens (Fig. 13) shows deep stra t i f ica t ion a t th e t ime of appearanceof the permanent lumen. The ant igens are to persis t in the st ra t i f ied squamonsepi the l ium of the subsequent s tages. The epi the l ia of the gastrointestinal tract,primi t ive and resembl ing tha t of the esophagus, a re r ich in ant igens (Figs.16 and 22) ; the la t te r , however , a re to wane and disappear upon fur ther de-velopment of the mucosae of the var ious segments, each proceeding a t i t sown pace . Thus, the mucosae of the body of the stomach (Figs. 16 to 19) , ofthe pylorus, and of the smal l in test ine rapidly lose the i r ce l l wal l ant igen a t35 to 40 rnm CR, whi le the colon and the rec tum fol low a t 45 to 60 mm CR.In each case the ant igen loss coincides wi th the assumpt ion of the specia l izedfunct ion of mucus secre tion.The epi the l ia of the parenchymatous organs der ived from the smal l boweldemonst ra te l i t t le or no ant igen. Thus, the pancreas manifests varying, ra thersmal l amounts, to be lost upon fur ther development of the gland (22, 23)while the liver i s f ree of the ant igens, even in the ear l iest avai lable specimens(Fig. 3).The trackea and m ain broncki are l ined by a pr imi t ive epi the l ium resembl ingthe digest ive t rac t and are equal ly wel l equipped wi th the ant igens (Fig. 13) .Those of the t rachea persis t throughout fe ta l l i fe , a lbe i t wi th grea t a t tenuat iontowards the end of tha t per iod, and are charac ter ized by scarc i ty and incon-stancy in postnata l l i fe . A unique pecul iar i ty of the developing bronchia l t reeis the consistent gross preponderance of the A,B ant igens over the H ant igen.At the sam e t ime, a reverse re la t ionship h olds for the ant igens of the pulm onarycapi l la ry endothel ium: the la t te r s ta ins poor ly wi th the ant i-A,B reagen ts andbri l l iant ly wi th the Bo mb ay conjugate (Figs. 14 and 15) . The bronchia l ant i -gens are not found af ter the 85 mm CR stage (12 weeks OA).

The bladder and the urogenital sinus are l ined by an epi the l ium which a t theear ly stages i s ident ica l wi th tha t of the hindgut f rom which they were ear l ie rpar t i t ioned off by the c loacal septum. The ce l l wal l ant igens are accordinglyvery w ell represented here . Thei r perm anence i s in agreement wi th th e genera lendow ment w i th the ant igens, of the st ra t i fied epi the lia, in th is ease the t ransi -t ional epi the l ium of the bladder (Fig. 26) and the st ra t if ied squam ous epi the l iumof the vest ibule and the lower vagina .



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510 A~ B, AND H ANTIGENS IN HUMAN :EMBRYOSThe mesonephros sh o ws b r i l l i an t o u t l i n in g o f t h e co l l ec ti n g t u b u l es a n d o f

t h e m a i n d u c t s . T h e a n t i g e n s c o u l d b e f o u n d a s l o n g a s t h e o r g a n r e m a i n e diden t i f iab le; i.e., u p t o 5 8 m m C R ( 1 0 t h w e e k O A ) . V e s t i g e s o f t h e d u c t a ls y s t e m i n t h e a d u l t f e m a l e (e.g. t u b u l es o f Ko b e l t ) sh o wed p er s i s t en ce o f t h ean t i g en s i n o ccas io n a l ce l l s . S imi l a r l y , t h e ep id id y mis o f n ewb o rn s sh o wedo ccas io n a l s t a i n ab l e ep i t h e l i a l ce l l s . Th e ep i t h e l i u m o f the metanephric ductan d i t s r ami f i ca t i o n s s t a i n u n i fo rm ly b r i l l i an t l y i n t h e ea r l i e s t emb ry o s (F ig .2 4 ). T h e a n t ig e n s r e m a i n p e r m a n e n t t h r o u g h o u t l i fe . N o t e w o r t h y a r e t h e p e r -s i s t en ce an d co n s i s t en t l y l a rg e amo u n t s o f ce l l wa l l an t i g en s i n t h e ep i t h e l i u mo f t h e co l l ec t i n g t u b u l es (F ig . 2 5 ) , t h e o n ly s imp le ep i t h e l i u m in l a t e f e t a l an dex t r au t e r i n e l i f e t h u s en d o wed .

I n the Mallerian duc ts t h e ep i t h e l i u m co n s i s t en t l y ev in ces t h e p resen ce o ft h e a n t ig e n s . N o c o r r e la t i o n w a s o b t a i n e d b e t w e e n t h e a m o u n t s o f t h e M t il -l e r i an an t i g en s an d t h e sex o f t h e i n d iv id u a l . Th e an t i g en s a r e fo u n d o u t l i n in gth e ep i t h e l i u m o f t h e l a rg es t sp ec imen i n t h i s se r i es an d a r e seen i n n eo n a t a l ,b u t n o t i n a d u l t, e n d o m e t r i a . T h e gonads, includ ing the germ cel l s , are devoido f an t i g en s .

T h e adrenal cortical ep i t h e l i u m i s co n s i s t en t l y n eg a t i v e (F ig . 2 4 ) , b u t t h ep o ss ib i l i ty t h a t t h e an t i g en s w ere p resen t d u r in g ea r l i e r s t ag es can n o t b eex c lu d ed . Ce ll s o f t h e ch ro m af f i n sy s t em sh o w n o a n t i g en s .B. Water-Soluble Ant igens

Th ese an t i g en s , b o rn e i n t h e sec re t i o n s o f the salivary glands (F ig . 2 0 ) an do f th e m u c o s a o f the gastrointestinal tract (F igs . 17 to 19) , f i rs t appear at 35 to4 0 m m C R ( 8 w e e k s O A ). T h e y a r e d e m o n s t r a b l e i n the submaxillary, sublingual,and parot id g lands as so o n as t h e l u men s i n t h e p r imo rd i a b eco me d i sce rn ib l e3A t t h e s a m e s t a g e , the py lorus b eg in s t o sec re t e l a rg e amo u n t s o f an ti g en s , w i ths m a l l e r a m o u n t s f o u n d i n the body o f the s tomach and in the goblet cel l s o f th esm all bow el; the duodenum c o m m e n c e s t h e s e c r e t i o n o f t h e a n t i g e n s r a t h e rl a t e , a t 6 5 m m C R . The large bowel seems t o as su me an t i g en i c sec re t i o n a t 4 5m m C R , w i t h the rectosigmoid e n t e r in g l a st , a t 5 8 m m C R .

Th e g o b l e t ce l l s an d smal l mu co u s g l an d s o f the respiratory tract h a v e n o tb een t r aced i n d e t a i l . Th e p resen ce o f g o b l e t ce l l s an d o f su b mu co sa l g l an d ssec re t i n g t h e an t i g en s h as b een eas i l y o b se rv ed in sp ec imen s ab o v e 8 0 m m CR .

The pancreas (F ig . 2 2 ) r emain s f r ee o f t h e sec re t ed an t i g en s u n t i l t h e 1 0 02The nature of the salivary and ga strointestinalsecretionshas not be en investigated beyondelucidating positive staining with PA S. It is noteworthy that both th e submaxillary and thesublingual glands, destined to secrete large proportions of m ucus, as w ell as the parotid, des-tined to serous secretion, are indistinguishab le both morphologicallyand with respect to theantigen content of their secretions. But, w hereas he antigens persist in the mucouselementsofthe mixed glands throughout life, they disappear from the secretion of the parotid shortlyafter birth.



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A R O N E. S Z U L M A N 51 1to 125 mm CR stage . Commencing secre t ion i s i l lust ra ted in on e of the la rgestspecimens of this series (Fig. 23).An t igen p re suma bly exc re t ed i n the urine and accre ted on the renal ca lyceal -pelvic epi the l ium has been ob served in severa l cases, the young est 40 mm CR .This reca l l s the appearance seen in postnata l mater ia l in secre tor individuals(1, 2).

The water-soluble ant igens cont inue to be secre ted dur ing subsequent fe ta ldevelopment and dur ing ext rauter ine l i fe by a l l the organs descr ibed. Twofetuses, 45 mm CR each (Tab le I ) show ed lack of the secre t ion-bound ant igensin the salivary glands (Fig. 21) with no significant diminution of those of thegast rointest ina l t rac t . These individuals are presumed to be non-secretors,especia lly s ince each had one non-secre tor parent . The problem of non-secret ionin int rauter ine l ife remains to be studied fur ther .

DISCUSSIONEpithel ial Cel l Wall A nt igc ns .--Th e mo st notew orthy f inding in this invest iga-

t ion i s the widespread dist r ibut ion of the epi the l ia l ce l l wal l ant igens ear ly inint rauter ine l i fe and the i r subsequent order ly recession. The survival of theant igens be yon d the 12th w eek OA, 70 to 80 mm CR , i s rest r ic ted to s t ra t i fiedor s imple confining epi the l ia ( in tegument , esophagus, lower ur inary t rac t ,vagina) , whi le epi the l ia proceeding to fur ther morphologica l and funct ionalcomplexi ty (gast rointest ina l t rac t , lung, thyroid, p i tui ta ry) undergo ant igenicloss in a manner and a t a s tage pecul iar to each t i ssue . Thus, the recession o/the cdl wall antigens takes place in close coincidence with recognizable stepsin histo-di f ferent ia t ion and commencement of mucous secre t ion in the respec-t ive segments of the gast rointest ina l t rac t ; i t occupies a br ief span, the 12thweek OA, dur ing which the thyroid begins to demonst ra te def ini te ac ini andis reported to commence speci f ic funct ion by accumula t ion of iodine (17) .The behavior of the ant igens in the ce l l s of the anter ior lobe of the pi tui ta rypresents a complex problem not least due to the gradualness of the changesand the mul t ipl ic i ty of the ce l l types involved (18) and has not been studiedin suff ic ient de ta i l . Li t t le i s known about pi tui ta ry funct ion dur ing ear ly fe ta ll i fe ; the demonst ra t ion of smal l amounts of ext rac table growth hormone in agland from a 12 weeks OA (85 mm CR ) fe tus (19) , however , serves to indica tethe p resence of funct ional ac t ivi ty in a gland in w hich ant igen loss is comple te .

The st r iking f reedom from ant igens on the par t of the l iver , the adrenalgland (cor tex) , and the nervous system is compat ible wi th the i r morphologi -cMly advan ced s ta te w hen f i rs t seen a t the 18 to 35 mm (5 to 8 weeks OA)stage and i s expl icable by assuming tha t these ce l l systems have a l readyundergone ant igenic loss. In the adrenal , the morphologica l advancement andextraordina ry vas cular i ty are para llel led by highly specif ic enzyma t ic compe-tence , demonst ra ted by biochemical techniques (20) in fe tuses f rom 40 mm



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51 2 A~ B, AND H ANTIGENS IN HUMAN E~BROSCR o n ; i n t h e l i v e r, ch o l es t e ro l sy n th es i s h as b een d em o n s t r a t ed a t 5 week sOA (2 1 ) .Th e v i r t u a l l y u n iv er sa l p r ev a l en ce o f t h e ep i t h e l i a l ce l l wa l l an t i g en s ea r l yin d ev e lo p men t l ead s t o t h e q u es t i o n as t o t h e i r f u n c t i o n i n emb ry o n a l l i f e .S in ce t h ey a r e s i t u a t ed a t t h e ce l ls ' su rf ace , t h e p o ss ib il i t y mu s t b e en t e r t a i n e dth a t t h e y a r e t h e se ro lo g i ca l r ep resen t a t i v es~ of a sui gener is b lo o d g ro u p su b -s t an ce co n t r i b u t i n g t o t h e mo lecu l a r p ro f i l e o f t h e ce l l wa l l an d p l ay a p a r t i nsuch proces ses as speci f ic cel l - to -cel l con tac t (22 , 23) o f especial s ign i ficancein em b ry o g en es i s (24 ) . Th e wan in g o f t h e an t i g en s s i gn i fi e s e i t h e r a ch an g e i nth e b lo o d g ro u p macro mo lecu l e o r i t s d i sap p earan ce an d co in c id es wi th t h eswi t ch in g b y t h e ce ll s t o m o re sp ec i f ic met a b o l i c ac t i v i t i e s , r e sp o n s ib le fo rr e c o g n i z a b l e s t e p s i n m o r p h o l o g i c a l a d v a n c e m e n t a n d / o r c o m m e n c e m e n t o ffu n c t i o n a l ac t i v i t y .

Wh i l e t h e mu co p o ly sacch a r id e n a tu re o f t h e b lo o d g ro u p su b s t an ces h as b eend emo n s t r a t ed fo r t h e wa t e r - so lu b l e en t i t i e s d e r i v ed f ro m sa l i v a , g as t r i c j u i ce ,an d mu c in o u s o v ar i an cy s t ( 1 1 , 1 2 ) , n o th in g i s k n o wn co n cern in g t h e wa t e r -i n so lu b l e su b s t an ces o f t h e ep i t h e l i a l an d en d o th e l i a l ce l l wa l l s , s av e fo r t h ese ro lo g i ca l i n d iv id u a l i t y o f t h e r eac t i v e g ro u p in g s . Th a t t h e re a r e d i f f e r en cesb e tween t h e u n d er ly in g macro mo lecu l es o f t h e v a r i o u s k in d s o f su b s t an ces i ssu g g es t ed b y t h e f i n d in g t h a t t h e b lo o d g ro u p su b s t an ce i so l a t ed f ro m e ry th r o -cy t es i s ch emica l l y s i g n i f i can t l y d i f f e r en t f ro m th a t o f t h e sec re t ed mu co id s(25).The Signi f icance of the Embryonal Antigens in Fetal-Maternal Relationships.- - A l a rg e b o d y o f s t a ti s t i ca l ev id en ce ex is t s p o in t i n g t o t h e se l ec t i v e i n t r a -u t e r i n e l o ss o f p ro g e n y cau sed b y f e t a l -m ate rn a l i n co m p at ib i l i t y wi th in t h eABO sy s t em ( r ev i ewed i n re f e r en ces 26 , 2 7 ) , t h a t i s , t o l o ss o f g ro u p A,B co n -cep tu ses , wh o se an t i g en s ( i n h er i t ed f ro m th e i r f a t h e r s ) a r e h o mo lo g o u s wi thex i s t in g ma te rn a l an t i -A ,B i so ag g lu t in in s . Th e ea r l y ap p earan ce , l a rg e amo u n t s ,an d t h e s t r a t eg i c d i sp o s i ti o n o f t h e ce l l wa l l an t i g en s h e re in d e sc r i b ed p ro v id ean i n s ig h t i n to t h e p ro cess l ead in g t o t h e l o s s o f i n co mp at ib l e emb ry o s b yrev ea l i n g th ese an t i g en s as p o t en t i a l t a rg e t s fo r mat e rn a l a n t i b o d i es ea r l y i np reg n an cy .* Th e im p o r t an ce o f t h e w a t e r - so lu b le an t i g en s i s p r esu m ed t o l i ei n t h e i r ab i l i t y t o p en e t r a t e i n to t h e f e t a l p l asma (2 8 ) an d t h u s t o se rv e as ab u f f e r ag a in s t ma t e rn a l an t i -A ,B i so ag g iu t i nin s . I t i s si g n i f ican t in t h i s co n -n ec t i o n t h a t t h e m ajo r i t y o f ab o r t i o n s , p e r fo rce i n c lu d in g t h o se d u e t o AB Oin co mp a t ib i l i ty , t ak e p l ace ab o u t t h e 8 th we ek OA, fo l l owin g t h e d e a th o f

3That the oligosaccharidicside chains imparting the A BH specificities hem selves are un-likely to be essential n any universal reaction or process s im plicit in the existence of individ-uals devoid of one (group A or B), two (group O), and all three (Bomba y) of the markers.4The notion that the ABH antigens so well established at 18 mm a ppear ev en earlier hasbeen borne out by finding he A antigen in a 5 m m specimen (27 days OA) studied at the timeof writing of the present com municationand to be published later.



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ARON E. SZULMAN 513

t h e emb ry o (2 9 ) , a t a s t ag e we l l b e fo re t h e p ro t ec t i v e b u f f e r i n g ac t i o n o f t h ewate r - so lu b l e su b s t an ces co u ld co me in to p l ay .

SUMMARYT h e A B H a n t i ge n s h a v e b e e n m a p p e d o u t i n t h e t is s u e s o f e m b r y o s a n d

fe tu ses , 1 8 t o 1 2 5 m m cro wn to ru m p l en g th , 6 t o 1 41 /~ week s o v u l a t i o n ag e .B o t h t h e H a n d A , B a n t i g e n s h a v e t h e s a m e d i s t r i b u t i o n , a n d t h e i r s p a t i a l

a n d t e m p o r a l p a r a l l e l o b t a i n s i n i n t r a u t e r i n e a s w e l l a s e x t r a u t e r i n e p h a s es o flife.

Th e ce l l wa l l an t i g en s a r e p resen t i n t h e i r max ima l d i s t r i b u t i o n i n t h ey o u n g e s t s p e c i m e n s a v a i l a b l e . T h e y o u t l i n e t h e e n d o t h e l i u m o f t h e c a r d i o -v a s c u l a r s y s t e m , a n d t h e c e l ls o f m o s t o f th e e p i t he l i a t h r o u g h o u t t h e b o d y .T h e e x c e p t i on s a re t h e l i v e r , t h e a d r e n a l , a n d t h e n e r v o u s s y s t e m , p r e s u m e dto h av e l o s t t h e ep i t h e l i a l an t i g en s a t s t ag es an t ed a t i n g t h e y o u n g es t sp ec imen sh ere d esc r i b ed .Th e an t i g en s o f t h e s t r a t i f i ed ep i t h e l i a ( an d o f t h e s imp le ep i t h e l i a o f t h eren a l co l l ec t i n g t u b u l es ) , t o g e th er wi th t h e en d o th e l i a l an t i g en s , a r e p e rman en tan d p er s i s t i n to an d t h ro u g h o u t ad u l t l i f e . A l l o th e r ce l l wa l l an t i g en s d i sap -p ear a t a t ime ch arac t e r i s t i c fo r each o rg an . Th e an t i g en i c r ecess io n co in c id esw i t h r e c o g n i z a b le s t e p s o f m o r p h ol o g i c a l a d v a n c e m e n t a n d o f t e n w i t h a s s u m p -t i o n o f f u n c t i o n b y t h e o r g a n c o n c e r n e d ; i t i s c o m p l e t e d a t a b o u t t h e e n d o ft h e f i rs t t r i m e s t e r o f p r e g n a n c y .

Th e sec re t i o n -b o rn e an t i g en s f i rs t ap p ear a t t h e 3 5 t o 4 0 m m s t ag e (8 week so v u l a t i o n ag e) i n t h e sa l i v a ry g l an d s an d i n t h e s t o m ach , t o b e fo l lo wed i n ac o n s t a n t s e q u e n c e b y t h e r e s t o f t h e g a s t r o i n t e s ti n a l t r a c t , r e s p i r a t o r y s y s t e m ,an d p an creas . Th e sec re t i o n o f t h ese an t i g en s p e r s i s t s t h ro u g h o u t l i f e .

Th e ea r l y p resen ce an d wid e d i s t r i b u t i o n o f t h e ce l l wa l l A ,B an t i g en sr e n d e r t h e m l i k e ly p o t e n t ia l t a r g e t s f o r m a t e r n a l a n t i -A , B a n t i b o d ie s i n h e t e r -o lo g o u s p reg n an c i es ; t h e ad v en t o f t h e w a t e r - so lu b l e su b s t an ces a t 8 week so v u l a t i o n ag e may b e p ro v id in g a b u f f e r sh i e ld in g t h e f e t a l ce l l wa l l an t i g en sb y m o p p i n g u p t h e m a t e r n a l i s o a g g lu t in i n s.

The aut hor wishes to thank members of the obstetrical staff at the Boston Lying-in Hospitalfor cooperation n obtaining material employed n this investigation; Miss P. A. Corcoran ofthe B lood Grouping Laboratory of Boston, for typing of many fetal and parental blood sam -pies; M rs. Josephine H. Bristol, M.A ., and Mrs. M artha Gelins, for highly exp ert technicalassistance.BIBLIOGRAPHY

1. Szulman, A. E., T he histological distribution of blood group substances A and Bin man, J. Exp. Med., 1960, 111 , 785.2. Szulman, A. E., T he histological distribution of blood group substances in m an asdisclosed by immunofluorescence. II . The H antigen and i ts relation to A andB antigens, J. Exp. Med., 1962, 115, 977.



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51 4 A, B, AND H AN TIGENS IN HUMAN EMBRYOS3. Oku, M., B lood groups in obstetrics and gynecology. II . B lood groups of imm aturefetuses, Jap . J . Obst. and Gynec. , 1930, 13, 472.4. Speiser, P., Uber die bisher jungste menschlische Frucht (27mm/22g) an derberei ts d ie Erbmerkm ale A, M, N , s , Fy (a+ ) , C, c , D, E , e , Jk , im Blu t fes tge-stell t werden konnten, Wien. klin. Woch. , 1959, 71,549 .5. Borenstein, S., and Israel, M., Agglutinogens in fetal erythrocytes, Proc. Soc.

Exp. Biol. and Med., 1942, 49,718.6. Constandoulkis, M., and Kay, H. E. M., A and B antigens of the human foetalery throcy te, Brit. J. Haematol., 1962, 8, 57.7. Dickgiesser, F., and Wildhagen, K., Untersuchungen fiber das Vorkomm en derBlutgruppensubstanz im fStalen Mekonium, Z. Immunit~itsforsch., 1952,109, 503.8. Wildhagen, K., and Kr ah, E., Zur Frage der embry onalen Entwicklung derBlu tgruppenmerkmale beim Menschen , Geburtsh. Frauenhe~k., 1952, 19., 744.9. Oku, M., Blood group in obstetrics and gynecology. III . Group specificity ofvarious o rgan cells in hum an fetus, .Tap. ]. Obst. and Crynec., 1930, 13, 524.10. HGgm an, C. F., Blood group antigens A and B determ ined by means o f mixedagglutination on cultured cells of hum an fe tal kidney, l iver, spleen, lung, hea rtand skin, Vox Sanguinie, 1959, 4, 319.11. K abat , E . A. , Blood Group Substances , Thei r C hemist ry and Imm unocbemist ry ,New York, Academic Press, Inc., 1956.12. Morgan, W. T. J. , A contribution to human biochemical genetics; the chemicalbasis of blood group specificity, (Croonian Lecture), Prov. Roy. Soc. London,Series B, 1960, 151,308 .13. Watkins, W. M., and Morgan, W. T. J. , Further observations on the inhibit ionof blood-group specific serological reactions b y simple sugars of kn own structure,Vox Sangulnis , 1962, 7, 129.14. G lynn, L. E., Holborrow, E. J. , an d Johnson, G. D., T he relationship of polym ersize and stflphation to th e h aptenic specificity of dextrans, J. Path. and Bact.,1954, 68, 205.15. McNeil , C., Trentelman, E. F., Kreutzer, V. O., and Fullmer, C. D., Aberrantsecretion of salivary A, B and H group substances in humans, Am. J . Cl in .Path., 1957, 28, 145.16. Thom as, D. B., Russel, P. M , and Yoffey, J. M ., Pat tern of haemopoiesis in thefoetal l iver, Nature, 1960, 187, 876.17. H odges, R. E., Evans, T. C., Bradbury , J. T., and K eettel , W. C., The accumula-tion of radioactive iodine by human fetal thyroids, J. Clin. Endocrinol. andMetab., 1953, 15, 661.18. Romeis, B ., H ypoph yse, in Handbuch der mikroskopischen Anatomie desMensc hen, (W. von Mollendorff, editor), Berlin, Springer-Verlag, 194 0, 6,tell 3, 136.19. Kaplan, S. L., and Grumbach, M. M., Non-specific inhibitors in serum and theimmunoassay o f hum an grow th hormone, J. Clin. Endocrlnol. and Metab.,1962, 22 , 1153.20. V illee, D. B., Engel, L. L., Loring, J. M., and Villee, C. A., Steroid hy droxyla -tion in human fetal adrenals: formation of 16 a-hydroxyprogesterone, 17-hydroxy progestero ne and deoxycorticosterone, Endocrinology, 1961, 60, 354.



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a x o N v.. s z ~ N 5 1521. Dav is, M. E., Plotz, E. J. , LeRo y, G. V., Gould, R. G., and W erbin, H., H ormone sin hum an reprod uction. I . Me tabolism of progesterone, A m . J . O b s t. a n d C , n e c . ,1956, 72, 740.22. W eiss, P., Cell contact, I n t erna t . R ev . Cy to l . 1958, 7, 39.23. Moscon a, A. A., Analysis of cell recombinations in experimental synthesis of tissues

i n v i t ro , J . Ce l l . and Comp. P hys io l . , 1962, 60, suppl. 1, 65.24. Holtfre ter, J. , Significance of the cell me mb rane in em bryonic processes, A n n .N e w Y o r k A c a d . S c . , 1948, 49, 709.25. Ko~cielak, J. , an d Zakrzewski, K ., Substances from eryth rocy tes of blood group AN a t u r e , 1960, 187, 516.26. Chung, S. C., and Morton, N. E., Selection at the ABO locus, A m . J . H u m a nGen., 1961, 18, 9.27. Lev ene, H., a nd Rosenfield, R. E ., ABO incom patibil i ty, i n Progress in MedicalGenetics, (A. G. Steinberg, editor), New York, Grune and Stratton, Inc.,1961, 120.

28. H ostrup , H., A and B blood group substances in the serum of the newborn infantand the foetus, V o x S a n g u i n is , 1963, 8, 557.29. He rtig, A. T., an d Sheldon, W. I-I., Minim al cri teria required to prov e prima faciecase of traumatic abortion or miscarriage, A n n . S u r g . 1942, 117, 596.



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516 A~ B~ AND H ANTIGENS IN HUMAN EMBRYOS

EXPLANATION OF PLATESPLATE 59

Endothelium, ErythrocytesFIG. 1 . Hear t in te ra t r i a l sep tum, 24 mm embryo , g roup A; an t i -A conjuga te .Well s taining endothel ium, desqu am ated in places. 170.FIG. 2. Ventr icular outf low tract and ar ter ioventr icular valve, 20 mm embryo,

group A; B om bay conjuga te . Iden t i ca l p ic ture ob ta ined wi th an t i -A reagent s . Con-t inuous layer of l ining end othd ium out l ined by ant igen. 170.FIG. 3. Sinus venosus and hepat ic s inusoids, 18 mm embryo, group A; Bombayconjugate. Ident ical picture obtained with ant i -A reagents (except for more dist inctstaining of hemopoiet ic cel ls) . Feeble but discernible s taining of the endothel ium ofthe he pat ic s inusoids. N ote the com plete out l ining of individual end othdia l cel ls in thesinus venosus. 170.FIG. 4 . Liver and hemopoie t i c ce ll s, embryo 18 mm, group A; r ab bi t an t i -A se rumand an t i - r abbi t conjuga te . A h igh power v iew of c lus te r s o f immature e ry throcytes ,well out l ined by d ouble layer s taining, against the backgro und of negat ive l iverparenchyma. 300 .Integument

FIG. 5. Skin, junct ion of lower l imb with t ru nk, 18 mm em bryo, group A; Bom bayconjugate. Ident ical picture obtained with ant i -A reagents . Well out l ined st rat i f iedepi thel ium; basal layer well s tained. 300.FIG. 6 . Skin , same s i tua t ion as in F ig. $ , 40 mm embryo , g roup B; Bo mb ayconjugate. Ident ical picture obtained with ant i -B reagents . Thin, wel l s tainingstrat i f ied epi thel ium; negat ive basal layer . 300.



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(Szulman: A, B, and H antigens in human embryos)



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PLATE 60Pituitary Gland

F I G . 7 . P i t u i t a r y g l a n d , g e n e r a l v i e w , 3 5 m m e m b r y o , g r o u p B , B o m b a y c o n j u g a t e .T h e e p i t h e l i u m p r o l i f e r a t e s i n s o l id m a s s e s i n t o a v a s c u l a r m e s e n c h y m a l s t r o m a . T h el u m e n o f R a t h k e ' s p o u c h i s c o m p r e s s e d t o a p o t e n t i a l s p a c e e x c e p t w h e r e i t w i d e n s , a tt h e r i g h t e x t r e m i t y o f t h e p a r s p o s t e r i o r . T h e e p i t h e l i u m i s s p e c i f i c a l ly o u t l i n e d , a s a r et h e s m a l l v e s se l s o f t h e m e s e n c h y m e i n t h e c e n t e r ; t h e c o n t r a s t w i t h t h e n o n - s t a i n i n gp a r s p o s t e r i o r i s w e ll d e m o n s t r a t e d . T h e c a r t i l a g e in t h e l e f t u p p e r c o r n e r s h o w s u p d u eto au to f luoresc ence . X 70 .

F I c . 8 . P i t u i t a r y , o n e " w i n g " o f a n t e r i o r l o b e , 2 7 m m e m b r y o , g r o u p A s t a i n e d w i t hr a b b i t a n t i - A s e r u m f o ll o w ed b y a n t i - r a b b i t c o n j u g a t e t o d e m o n s t r a t e t h e s t r i k i n gd e f i c i en c y o f A a n t i g e n . T h e e n d o t h e l i u m s h o w s t h e u s u a l a b u n d a n t A a n t i g e n . 1 7 0.

F I G. 9 . P i t u i t a r y p o s t e r i o r l o b e a n d p a r s i n t e r m e d i a , 3 5 m m e m b r y o , g r o u p B ,( f r om t h e s a m e c a s e a s F i g . 7) ; h u m a n a n t i - B c o n j u g a t e . T h e p a r s p o s t e r i o r i s d e v o i do f a n t i g e n s ; t h e p a r s i n t e r m e d i a s t a i n s f e e b l y w i t h t h e a n t i - B r e a g e n t ( c o m p a r e w i t ht h e b r i l l i a n t e n d o t h e l i u m o f v e ss e l s r u n n i n g a l o n g t h e p e r i p h e r y o f p a r s p o s t e r i o r ) .T h e o r i g i n a l l u m e n o f R a t h k e ' s p o u c h i s r e p r e s e n t e d b y t h e s p a c e i n l e f t u p p e r c o r n e r .X 170.

F I G . 1 0 . P i t u i t a r y a t a b o u t 2 5 w e e k s ' g e s t a t i o n , g r o u p 0 , B o m b a y c o n j u g a t e .T h e a n t e r i o r l o b e , u p p e r l e f t , h a s l o n g l o s t t h e a n t i g e n c o m p l e t e l y ; i t s ho w s t h e r i c hv a s c u l a r i t y o f a n e n d o c r i n e , a s d o e s t h e p a r s i n t e r m e d i a ( b e l o w a n d t o t h e r i g h t o f t h er e s i d u a l l u m e n o f R a t h k e ' s p o u c h i d e n t i f i a b l e a s a n a r r o w d i a g o n a l s l i t ) . T h e p a r sp o s t e r i o r , l o w e r r i g h t , s h ow s c o m p a r a t i v e l y p o o r v a s c u l a r i t y . 1 7 0.Thyroid , Thymus

F I G . 11 . T h y r o i d , t h y m u s , t r a c h e a , a n d e s o p h a g u s , 1 8 m m e m b r y o , g r o u p A ;h u m a n a n t i - A c o n j u g a t e . T h e t h y r o i d i s s ee n a s a s o l i d , sp e c i f i c a l ly s t a i n i n g c r e s c e n t o fe p i t h e l i u m i n t h e l o w e r p a r t o f t h e p h o t o g r a p h , l y i n g i n f r o n t o f t h e t r a c h e a a n d t h ee s o p h a g u s . T h e t h y m u s i s r e p r e s e n t e d b y t h e t w o a n l a g e n s e e n a s t w o s m a l l r o u n dm a s s e s o f s p e c if i c a l l y s t a i n e d e p i t h e l i u m o n e a t e a c h e x t r e m i t y o f t h e a r c o f t h e t h y r o i d .T h e p r i m i t i v e t r a c h e a a n d e s o p h a g u s d e m o n s t r a t e w e l l t h e s p e c i fi c s t a i n i n g o f t h e i rep i the l ia . 70 .

F I G . 1 2. T h y r o i d , f e t u s 1 50 m m C R ( m i d - p r e g n a n c y ) , g r o u p 0 ; B o m b a y c o n j u g a t e .A h i g h p o w e r v i e w t o i l l u s t r a t e t h e a b s e n c e o f a n t i g e n f r o m t h e c e l l w a l ls o f t h e a c i n i ;p e r i a c i n a r c a p i l l a r i e s w e ll v i s u a l i z e d b y v i r t u e o f s p e c if i c e n d o t h e l i a l s t a i n i n g . ~ ( 2 00 .



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PLATE 61Respiratory System

F IG . 13 . M e d i a s t i n u m , 1 9 m m e m b r y o , g r o u p 0 ; B o m b a y c o n j u g a t e . T h e b i f u r c a -t i o n o f t h e t r a c h e a ( t w o c e n t r a l t u b e s ) a n d t h e r i g h t m a i n b r o n c h u s ( l o w e r r i g h t o fp h o t o g r a p h ) a r e w e l l v i s u a l i z e d b y v i r t u e o f s p e c if i c o u t l i n i n g o f t h e e p i t h e l i u m .T h e e s o p h a g u s w i t h i t s w e l l s t a i n i n g e p i t h e l i u m i s s e e n b e lo w t h e t r a c h e a l b i f u r c a t i o n( c o m p a r e F i g . 1 1 ) . T h e a o r t a , i n t h e l e f t l o w e r c o r n e r , a n d t h e a t r i a a t t h e u p p e r e d g eo f t h e p h o t o g r a p h d e m o n s t r a t e t h e u s u a l e n d o t h e l i a l s p e c if i c s t a i n i n g . 7 0 .

F I GS . 1 4 a n d 1 5. P r i m i t i v e l u n g fr o m t h e s a m e 1 8 m m e m b r y o , g r o u p A . I n F i g .1 4, n o t e t h e a b u n d a n c e o f A a n t i g e n i n t h e b r o n c h i a l e p i t h e l i u m a n d i t s s c a r c i t y in t h ec a p i l l a r y e n d o t h e l i u m ( h u m a n a n t i - A c o n j u g a t e ) . F i g . 1 5 s h o w s t h e s c a r c i t y o f Ha n t i g e n i n t h e b r o n c h i a n d l a r g e a m o u n t s o f i t i n t h e c a p i l l a r i e s ( B o m b a y c o n j u g a t e ) . 70.Stomach (Corpus)

F IG . 16 . L i n i n g e p i t h e l i u m , e m b r y o 1 8 m m , g r o u p A ; B o m b a y c o n j u g a t e ( i d e n t i c a lp i c t u r e o b t a i n e d w i t h a n t i - A r e a g e n t s ) . E p i t h e l i u m w e l l o u t l i n e d b y a n t i g e n ; n o t eu n i f o r m , u n d i f f e r e n t i a t e d c h a r a c t e r o f t h e m e m b r a n e . X 1 7 0.

F I G . 1 7. M u c o s a i n a n o l d e r s p e c i m e n , 40 m m C R , g r o u p B ; h u m a n a n t i -B c o n j u g a t e . N o t e s t r u c t u r a l a d v a n c e m e n t o f t h e m u c o s a ; t h e c e l l w a l l a n t i g e n i sw a n i n g r a p i d l y a n d s e v e r a l b l e b s o f s e c r e t i o n b e a r i n g t h e a n t i g e n h a v e a p p e a r e d . 170.

F I G . 1 8 . M u c o s a f r o m f e t u s 7 0 m m C R , g r o u p 0 ; B o m b a y c o n j u g a t e . S e c r e t i o n o ft h e m u c u s - b o r n e a n t i g e n i s w e l l i l l u s t r a t e d ; t h e c e l l w a l l a n t i g e n i s a b s e n t ; s o m e m u c u sc o a t i n g o f e p i t h e l i a l c e l ls h a s t a k e n p l a c e . 3 0 0.

F I G . 1 9 . G e n e r a l v i e w o f m u c o s a o f s e c r e t o r t y p e , f e t u s 1 1 5 m m C R , g r o u p A ;r a b b i t a n t i - A s e r u m a n d a n t i - r a b b i t c o n j u g a t e . A b u n d a n t m u c u s - b o r n e a n t i g e n ; n os t a i n i n g o f c e l l w a l l a n t i g e n s . 7 0 .



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PLATE 62Salivary Glands"

FIG. 20. Submaxill ary gland, fetus 40 mm crown to rump, group B ; huma n anti-Bconjugate. (Similar picture obtained with the Bombay conjugate.) Commencingsecretion rich in antigen. X 170.

FIG. 21. Sub maxillary gland, fetus 45 mm CR, group 0; Bo mbay conju gate. Nosecreted antigen s demonstrated in serial sections of this gland and of the p arot id;this case is presumed to be a non-secretor. >( 170.Pancreas

Fro. 22. The formation of the gland by an outgrowth from the du odenum, 18 mmembryo, group A; hum an an ti-A conjugate. Note the well marked cell-bound antig enin the duodenum and in the pancreatic duct, and its absence from the tubules.X 70.

FIG. 23. Commencing secretion of antigens in some pancre atic acini in a fetu s 110mm CR, group 0; Bombay conjugate. 300.Kidney, Adrenal, Bladder

FIG. 24. Anlage of the permanent kidney and adrenal, embryo 27 ram, group A;human anti-A conjugate. The early kidney shows the well established branching ofthe ureteral bud. Note the abundance of the cell wall antigen of the epithelium, afeature to persist into adult life from the collecting tubules down to the ureter.

The adrenal is seen between the stomach (above) and the metan ephros (below).There is no cell wall antigen in the parenchymal elements; there is remarkable vas-cularity visualized due to endothelial specific staining. The vessel at the right edge ofthe photograph is the aorta. 30.

FIG. 25. Kidn ey, fetus 65 mm crown to rump, group A; ra bbit anti-A serum andanti-rabbit conjugate. Continued abundance of antigen in the branching ureteralbud, as it forms the pelvis, calyces, and collecting tubules; other tubules devoid ofantigen. The glomeruli stain by virtue of endothelial antigen. >( 70.

FIG. 26. Urin ary bladder, fetus 115 mm CR, group A; huma n anti-A conjugate.Sharp out linin g of cells of trans itiona l epithelium. The d esquamated cells in the lumentend to show cytoplasmic staining. X 70.



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