Distribucion y Expresion Tisular de La Semelogelinas

7/29/2019 Distribucion y Expresion Tisular de La Semelogelinas

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journal of A nd ro logy , V ol. 17 , N o . 1, January /February 1996Copyr igh t u American S oc ie ty o f A nd ro lo gy

D istr ib u tion and T issu e E xpress ion o fSem enoge lin I an d II in M an as D em on stra ted byIn S itu H ybr id iza tion and Im munocy toch em istryANDER S B JARTELL ,* JO HAN M ALM ,t CH R IST INA M OLLER , M ATS GUNNAR SSON ,*AK E LU ND WA LL ,f AND HAN S L IL JA tF rom the D epartm en ts o f *U ro lo gy jC lin ica l C hem is try , a nd Patho log y, U n ive rs ity H osp ita l , M alm # {246} ;an d th eD epartm en t o f U ro lo gy , U nive rs ity H o sp ita l, Lund , U n ive rs ity o f Lund , Swede n .

A B S T R A C T : S em en o ge lin I a n d II ( S g l, S g ll) a re tw o se pa ra te g en ep ro du c ts o f ch rom os om e 2 0 w ith ex te ns ive (-8 0% ) ide n tity in pri -m ary s tru c tu re . T h e y a re m a in ly re spo n s ib le fo r Im m e d ia te ge l fo r -m a tio n o f fre sh ly e jac u la te d se m e n . D eg ra da tio n o f S g l an d S g ll isd ue to th e p ro te oly tic a ctio n o f p ro sta te -s pe cific an t igen (P S A ); itre su lts w ith in 5 -15 m inu tes in liq ue fac tion o f s em e n an d re le ase o fp rog re ss iv ely m otile sp erm atozo a. B y m ea ns o f cD N A c lo n in g an dN o rthe rn b lo ts , S g l a n d S g lI tra n sc rip ts ha ve pre v iou s ly b ee n s h o w nto be a bu nd a n t in hu m a n s em ina l ve s ic le s , bu t S g ll a lo ne is su g -ge s te d to b e e xp re sse d a t low le ve ls in the e p id id ym is . T o cha ra c -te rize th e e xp ress io n an d tissu e d is tr ib u tion o f S g l an d S gll in g re ate rde ta il, w e p ro d uce d m on o c lo na l Im m u n og lo bu lin G s ( lg G s) fo r im -m uno cy to che m is try (IC C ) an d s pe c ific [M S]-, d igo x ig e n in -, o r a lka lineph os ph a ta se -la be le d 3 0-m e r a n tis en se p rob e s to S g l a n d S g ll fo rin s itu hy brid iza tion (IS H). Immu n o c y to c h e m ic a l s ta in in g fo r b o th S gla n d S g ll, a n d IS H de tec tion o f b o th S g l a n d S g ll tran sc rip ts , w eredemons tra ted in th e cy top la sm o f se m in a l ve s ic le e p ithe liu m . IS Hs ho w e d S g ll a lon e to b e e xp re ss ed in th e e p ithe lium o f th e ep id idyma l

c au da . N e ith e r IC C no r IS H y ie ld e d a n y e v id e n ce o f S g l o r S g lle xp re ss io n In cap u t o r co rp us e p ith e liu m o r In a ny s tro m al c e lls o fth e ep id idymis . C ons is te n t w ith ou r p rev io us find in gs u sin g p olyc lo na llg G , m o no c lon a l an ti-S g l/S g Il lg G s id en tif ie d ep ito pe s o n th e p o s-te rio r he a d , m idp iece , a n d ta ll o f e jac u la te d sp e rm a to zo a . S p erm a-to zoa in the ep id idym a l ca ud a w ere a lso im m un o rea c tiv e , bu t th o sein the c ap u t o r co rpu s reg ion o f the ep id id ym is a s w e ll as th ose inthe te s tis w e re ne g a tiv e . A s sh o w n by IC C , ne ith e r S g l no r S gll w eree xp res sed In the te s tis , th e p ros ta te , th e fe m a le g en ita l t rac t , oro th e r no rm a l hu m a n tis su e spe c im e ns . A lth ou g h th e s ign ifica nce o fS g a ttach m e n t to ep id id ym a l a n d e jac u la te d s pe rm a toz oa re m a in sto be es ta b lish e d , m o no c lo n a l an ti-S g lg G m ig h t p ro ve usefu l ines tab l ish ing th e o rig in o f se m ina l ve s ic le tiss ue com po n en ts in p ro s -ta te c ore b io ps ies o r o th e r b iopsy s p e c im e n s .

K e y w ords : S e m e n , spe rm a to zoa , fe r ti liza tion , S o u th e rn b lo t, o Il-go de o xyn uc le o tid e p rob e s .

J Andro l 1 9 9 6;1 7 :1 7-2 6

T h e e ja cu la to ry mix ing o f secre tio ns from the sem in alve s ic les and the p ro sta te re su lts in im m ed ia te fo r-

m a tion o f a g e l in w h ich sem enoge lin I and II (S g I andSgI I ) cons t i tu te the m ajo r g el p ro te in s (L il ja an d Laurel l ,1984 , 19 85 ). P ro g re ssive ly m oti le sp erm a tozoa are re-lea sed w hen the g el d isso lv es (i.e ., the e jacu la te liq ue fies )a s a resu lt o f p ro teo ly tic deg rad atio n o f th e g el p ro te in s(L ilja and L aure ll, 19 84 ; M cG ee and H e rr, 19 87 ). T h isd eg rad a tion is du e to the action o f p ro s ta te-spec ific an -t igen (P SA ) (L ilja , 1 98 5 ; L ilja e t a l, 19 87 ; M cG ee andH err, 1 98 7 ; L ee e t a l, 1 98 9 ), a m o st ab undan t p ros ta te-

Suppor ted by th e Sw ed ish M edical Resea rch Counc il (g ra nt p ro je ctn os. 1 3X -7 903 and 8660), th e F acu lty o f M edic ine a t L und Un iv ersity ,the R esearch F und and th e C ancer Research F un d a t M alm #{ 24 6} en era lHosp i ta l , th e Sw ed ish S oc iety of M edic ine (SLS), th e F ound ation fo rU ro logy R esearch in Malm#{246 } , the M agnu s B ergw all Foundat ion , th eFounda tion of C rafoord , and the F und ac ion F red erico S .A .

C o rre spond ence to: D r . Anders Bjar te l l , D ep artm en t o f U ro logy , U ni-versity H osp ita l, M alm #{246 }S -20 5 02 M alm #{246} , Sw ed en .

R ece ived fo r p ub lication Ju ly 5 , 1 995 ; accep ted fo r p ub lication S ep-tem ber 19 , 1 99 5 .

de r ived ser ine p ro tea se in th e sem in al flu id , w ith ex ten sives tru ctu ra l sim ila rity to the g land u la r k all ik re in s bu t w ithan enzym e action sim ilar to th a t o f chym o tryp s in (W a tte t a l, 1 98 6 ; A k iy am a e t a l, 1 987 ; L undw a ll and L ilja ,19 87 ; S cha lle r e t a!, 1 98 7 ; L ilja e t a !, 1 98 9 ; C hris ten sso ne t a l, 19 90 ).

T he -52 -kD a Sg I an d -70 -kD a Sg II a re p red om inan tse cre to ry p ro te in s from the sem ina l v es ic le s (L ilja e t a l,19 84 , L ilja an d L aure l, 1 98 5 ). T he se p ro te in s h av e m anyp rop ert ies in com m on w ith the sem in al ve sic le -specifican t igen (SV SA ) de scrib ed b y H e rr e t a ! (1 98 6). T he re fo rei t is lik ely th at th e m onoc lon a l an tibo dy M H S-5 used toiden tify SV SA m ay d e tec t S g I an d S gII. S g I is a sing le-cha in , no n -g lyco sy la ted p ro te in o f 4 39 am in o ac ids (L iljae t a !, 19 89 ). T he s tru ctu re o f Sg II is ve ry s im ila r (-8 0%iden tity in p r imary s tru ctu re), b u t it con ta in s 55 9 am inoac ids (L ilja and L undw all , 1992). SgI is sug ge s ted to beexc lu s ive ly exp ressed in th e sem in al v es ic le s (L ilja e t a!,19 89 ). T h e sem ina l v es ic le s a lso co ns ti tu te th e m a jo r se -cre tory orig in o f S gII, a lth ough S gII m RNA is a lso d e-tec ted a t low leve ls in th e ep id id ym is (L ilja an d L undw a ll,


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18 J ou rn al o f A nd ro lo gy J an u ary /F e br ua iy 1 99 6

1985) .

1992). Ep id idym a l ex pre ssio n o f SgII is fu rth er su ppo rtedb y the de tectio n of im m unoreac tiv ity to SgI/S gII lo calizedto the sec re to ry ep ithe lium of the ep id id ym is, in ad d itionto th e Sg I/S gII- imm uno sta ined ce lls in th e ep ithe lium o fth e sem in a l v es icle s (L ilja Ct al, 19 89) . M oreo ve r, th etis sue -specif ic exp re ssio n of these p ro tein s m ay b e pa r-ticu lar ly im portan t, becau se specif ic im m unosta in ing fo rb o th Sg I/S gII and SV SA has been loca lized to the po s-te r io r pa rt o f th e head , m idp iece, and ta il o f e jacu latedsp erm a tozoa (H err e t a l, 1986 ; L ilja e t a l, 198 9) . P rev io uss tud ie s have been lim ited to the u se o f p o lyc lo na l rabb itim m unog lo bu lin G (IgG ) aga inst a 52 -am ino -ac id frag -m en t o f SgI (L ilja e t a l, 19 89 ). T his an tibod y c ross-reac tsw ith SgII du e to th e ex ten sive s tru ctu ra l s im ila r ity b e-tw een Sg I and SgII (L ilja and Lundw a ll, 199 2) . To cha r-ac te rize the tissue ex pre ssio n of SgI and Sg II in g reate rde ta il, pa rticu la r ly in the m ale gen ita l trac t, w e gen era tedan d cha racte riz ed m ono clo na l an tib od ies aga in s t pu rifiedS gl an d SgII fo r imm unocy to chem ical stu d ie s , an d w ep ro duced an tisense o lig odeoxynu c leo tide p robes spec ificto e ithe r S gI or Sg II transcr ip ts fo r use in d eta iled in si tuhyb rid iza tio n (ISH ) s tu d ie s .

M ate ria ls a nd M eth od sR e a g e n t sTh e S gI fra gm en t en compass i n g res idues 85-136 w as purifiedas ea rlier d esc r ibed (L ija an d Jeppsso n , 198 5 ; L ilja et a l, 1 989) .B io tiny la ted go at an t i- rabb i t IgG and b io tiny la ted horse an ti-m ouse IgG , no rma l m ouse and norm al rabb it IgG l, s trep tav i-d in -horse rad ish perox id ase (SA -H RP) con juga te , and norm alsh eep serum (N SS ) w ere ob ta ined from D akop atts A /S (G los-tru p , D enm ark ). D iam in obenz id in e (D AB ) an d Fast R ed , as w ellas V ec tab ond co atin g so lu tion fo r m icrosco p ic slide s, w e re sup -p lied b y V ec to r Labora to r i e s (Bur l ingame, California). Acetan-hydride an d tr ie thano lam in e w ere purchased from BDH L td .(P oo le , U K ), fo rm am ide and T riton X - 100 from M erck (D arm -stad t, G e rm any), M oun t-Q u ick A queous from D aido Sa ngyoCom pany , L td . (Jap an ), P ertex from H isto lab s (G o thenbu rg ,Sw eden) , Ilfo rd K -S au to rad iog raph y em ulsion from Ilfo rd Sci-en tific P roduc ts (C hesh ire , U K ), D ek to l D - 1 9 deve loper andX -A R5 film from Eastm an K odak Com pany (R o cheste r, N ewY ork), an d Age-F ix from A gfa-G aev er t A G (L everkusen , G er-m any). N ylon hyb rid iza tion filters (H ybond N ), rad ion uc leo tides[a -35S )dA TP (no . SJ 1304 ) and [y -32P ]ATP , an d a 5-end lab e lin gki t (no . 402 0) w ere from Am ersh am In terna tion a l (B uck ing -ham sh ire , UK ). T he d igox igen in (D IG ) o ligon uc leo tide tailingki t (no . 1 417 231 ) an d Ft,, fragm en ts o f an ti-D IG alka lin e pho s-pha tase (A P) con jug a te an d pro te inase K were supp lied by Boeh -rin ge r M annhe im GmBH (M annh e im , G erm any ). C hrom osp in -10 ge l-ex clu sion co lum ns w ere from Clon te ch L abora to rie s Inc .(Palo Alto , C alifo rn ia) , and T4 polynuc leo tide k inase w as fromPharm ac ia (U ppsa la , Sw eden) . A ll o th e r reagen ts w ere o f re agen tgrade an d pu rchased from S igm a C hem ica l C om pany (S t. L o u is ,Missour i ) .

T is s ue S p e cim e n sFor ISH and im munocy tochem istry ( ICC ) s tud ies, tis su e spec-im en s w ere o b ta ined a t su rge ry . S em in al vesicle s and p ro stateg lan ds w ere rem oved a t cysto pros ta tec tom y from 10 pa tien ts(age 60-7 4 y ears ) w ith cancer o f the ur in ary b lad der. Spec im ensfrom the testis, de fe ren t du cts , and th e ep id idym is w ere takena t o rch idec tom y as su rg ica l trea tm en t o f p ros ta tic cancer in 10pa tien ts (ag e 72 -81). In ad d itio n , the ep id id ym ides from twopa tien ts w ere d iv id ed lo ng itud ina lly to s tudy the reg ion a l d is-tribu tion of Sg I/SgII ep itopes in th is o rgan . T he prov is ions o fthe H elsink i D ecla ra tion regard ing the u se of hum an tissues w eres tr ic tly o bs er ve d.

W ith in 10 m inu tes after co llec tion , a ll spec im en s w ere im -m ers io n-fixed in B ou in s fixa tive or in 4% fo rm ald eh yde in phos-ph a te-bu ffered salin e (PB S), dehy drated , an d em bedded in pa r-a ff in . S ectio ns w ere cu t a t 3 -tim th ick ness an d m oun ted o nchrom e-a lum -coa ted (sub bed) slides fo r IC C (H u ang et a l, 19 83),o r V ec tabond -coa ted or 3 -am in oprop y ltrie thox ysilane (APES)-coa ted slide s (R en trop e t a l, 1986) fo r ISH . A rch iva l fo rm alin -f ixed p a ra ff in -em bedd ed spec im ens w ere o b ta ined from th e D e-pa rtm en t o f P a tho logy a t th e U niversity H osp ita l, M alm #{246} . H e -m ato xy lin -eosin -sta ined sectio ns from all tissue specim en s w ereh is to pa tho log ica lly no rm al.

F re sh e ja cu la te d sperm atozoa from four vo lun tee r dono rs w ithno rm al spe rm a tog ram s w ere cen tr ifug ed at 8 00 x g fo r 10 m in-u te s and then w ashed tw ice in 0 .05 M sod ium phosp ha te , pH7 .4 , an d 0 .15 M N aC 1 to rem ove loo se ly bo und sem in a l p lasmacom ponen ts. T he sperm atozoa w ere recovered by centrifugat iona t 800 x g fo r 1 0 m inu tes a fte r each w ash ing cy cle , sm eared o nun trea ted s lid es , and sto red a t - 70#{176}C .or ICC , th e sp erm atozoaw ere dried ov ern igh t a t room tem pera tu re , f ix ed in ace ton e fo r10 m inu te s, an d a ir-d ried .

Ant ibod iesU sing prev ious ly desc ribed m ethods , m ono clo na l an tibo d ies w ereraised in B alb /c m ice aga inst S gI purif ied from hum an sem enco llec ted in a lka line bu ffer con ta in ing u rea to p reven t deg rad a -tion of the sem enoge lins (B orrebaeck an d Ey la r, 19 81 ; L aure lle t a l, 1 988). T h e purified SgI w as essen tially h om ogenou s asjudg ed from sod ium dodecy l su lfate po ly ac ry lam ide ge l elec tro -pho resis (SD S/PAGE ), the on ly con tam inan t be ing m in oram oun ts o f SgII. T he m ice w ere imm un ized w ith approx im ate ly15 ig pu rif ied S gI and g iven a booste r w ith the sam e am oun t o fan tigen a fte r 1 and 3 w eek s. B efo re fus ion of sp leen ce lls (l0 )w ith N S 1 m yelom a ce lls, the m ice w ere g iv en a booste r o f-V 100g an tigen i.p . o n th ree con secu tive day s. A so lid -p hase rad io -imm uno as say u sing m icro titratio n w e lls coa ted w ith a m ix tu reof S gI and SgII a s an tigen (1 b ig /w ell) w as used to iden tify pos itivec lones, a s p rev iously desc rib ed (L au re ll e t a l, 1 988 ). Pos i t ivec lones w ere subc loned th ree tim es by lim ited d ilu tion , ex panded ,and in jec ted i.p . in to pris tine -p rim ed B alb /c m ice. T he an ti-bod ie s w ere iso la ted b y io n -ex ch ange ch rom a tography on Q-Se -ph arose F ast F low , using standa rd p rocedu re s. T he iso typ es o fthe an tibo d ies w ere de te rm ined w ith a do ub le -an tibody sand -w ich en zym e-link ed imm uno sorb en t assay (EL ISA ) (Sugo e t a !,


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Barte ll e t a l T is su e E xp re ss io n o f S em e no ge lin I a nd !! 19Immunocytochemis t ry ( ICC)M urine m onoc lona l and po ly clo na l rabb it IgG s ag ains t S gI/S gIIw ere d etec ted u sing a SA -H RP con ju ga te an d DAB (E lia s e t a ! ,198 9) . To com pare imm unos ta in ing p atte rns ob ta in ed by d if -fe ren t IgG s , an d to com pare IC C w ith ISH , 3-tim ad jacen t sec -tio ns w ere m oun ted on sepa rate slide s. S g I/S g II ep itop es on theeja cu la ted spe rm ato zo a w ere d ete c ted by m onoc lon a l an t ibo d ie sag ain st S g I/S g II u sing the alk a line pho sph ata se -an ti-a lka linep hosp ha ta se (A PAAP ) techn ique with Fast R ed as the ch ro -m ophore (C o rd ell et a ! , 19 84) .

T he m ono c lo na l S gI/S gII IgG s w ere used a t a fin al concen tra-tio n of -25 ,zg /m l in ICC , and the p rev iously d esc ribed aff in ity -purified p o lyc lon al rabb it IgG s aga inst th e 52-am ino -ac id frag -m en t o f S gI (L ilja an d Laure l, 1 985 ; L ilja e t a1 , 198 9) w ere useda t a fin al con cen tra tion o f 1 2 g /m l.

P u rified no rm al m ouse and rab b it IgG 1 w ere used as rep lace-m en t fo r the m onoc lo na l and po ly c lona l S g an tib od ie s , re spec -tive ly , and they served as nega tive con tro ls in ICC . In an o the rp rocedure to ve rify th e sp ec if icity o f th e im m unosta in ing reac-tion s, the fragm en t o f S g I encom passin g re sid ues 85 -1 36 th atw as added to the a ffin ity -p u rified p o lyc lon al an tib od ie s in se ria ld ilu tio ns up to 60 tim es m o la r excess (-3 00 ig /m l) w as used .

O lig odeo xynuc leo tide P robesAn tisense o lig odeoxynu c leo tide s (3 0 -m ers) w ere sy n thesized o nan A pp lied B io system s 38 1A DNA syn th es ize r . T he p ro bes w erech osen to p rov ide an op tim a l n um ber o f m ism a tches b etw eenSg I and SgII: 5 -C CAA TCC CCC ATG AGA TCC ATG TFGGTG TC -3 com plem en ta ry to nuc leo tide s 1280-1309 of thehuman Sg I tran scr ip t (L ilja e t a l, 198 9) and 5-T A TG TGAC TC AC T GGA TTC CT G TTT GTA TC -3 com plem en ta ryto nuc leo tide s 1628-1657 o f the hum an SgII transc r ip t (L iljaan d Lundwal l , 1992). B o th p rob es w ere sea rch ed fo r sim ila ritie sw ith h um an m amm alian n uc leo tide sequences in G enB ank#{174 }(release 71 .0 ) using th e FASTA#{174 }p ro g ram . In add ition , an oh-god eoxy th ym id in e (o ligo -dT ) p rob e (3 0 -m er) w as syn the s izedan d u sed as a pos itive co n tro l in ISH . T he p ro bes w ere p urif iedby PAGE befo re use (S am brook e t a !, 1989).

Probe Labe l ingO ligo deoxynuc leo tid es w ere labe led w ith D IG us ing a 3 -endlabe ling k it fo r ta iling reac tion w ith D IG -il -dUTP and dTTP ,catalyzed by te rm ina l deoxy transfe rase (T dT ) accord in g to them anu fac tu rers p ro toco l.

O ligod eo xynuc leo tide s com plem en ta ry to SgI and SgII t ran -sc r ip ts , o u tlined as abov e, con tain ing 5 - term in al am in o group s,w ere a lso con juga ted to alkaline phosph a tase (A P ) an d sup p liedready fo r use b y R& D Sy stem s E urope L td . (A b ing don , O xon ,UK ) .

R ad ioac tive ISH prob es w ere lab eled w ith [a -3 5S ]dATP to aspec ific activ ity o f> 1 .5 x l0 co un ts pe r m in u te (cpm )4 ig p rob e,usin g a labe ling k it inc lud ing T dT as th e ca taly zin g enzym e.

Fo r Sou th ern b lo ts , o ligo deoxynuc leo tides w ere 5-end labe ledw ith [y -32P ]A TP and T 4 po lyn uc leo tide k in ase to a spec ific ac -tiv ity o f >1 08 cpm 4tg prob e.

Sou thern B lo tT he specificity of each of the SgI and SgII p robes w as ve rif iedby Sou th ern b lo t ex pe rim en ts , dem onstratin g tha t each proberecogn ized on ly the re spec tive cDNA clo ne u nd er str ingen t co n-d itio ns and p rod uced m in im a l c ross-hy brid izatio n sign a ls to th eo ther cDNA clon e . T h e E coR 1 inserts co d ing fo r the cDNAc lones o f Sg I (XSGLN- IV ) and of SgII (X V SRP -XX I) (L ilja et a! ,198 9 ; L ilja and Lundwal l , 1992) w ere e lectrop hore sed in 1%agarose g els , v acuum blo tted to H ybond N ny lon filte rs , an dhybrid ized to the rad ioactiv e p ro bes. Th e filters w ere rin sed an dw ashed in 2 x s tandard sa line c itra te (SSC ) an d 0 .1% SD S for3 0 m inu tes a t 4 5#{176 }Cnd 15 m inu te s a t 54#{176}Cnd exposed toKodak XAR -5 film for 30 m inu tes .

I n s it u H y br id iz at io n ( ISH )AP-con juga ted Probes -The p ro toco l fo r AP-con juga ted probes

w as b ased o n methods described by K iy ama et a ! (1 99 1) andrecently pub lished in de ta il (B ja rte ll e t a !, 1 993 ).

D IG -la be le d P ro bes-T he u se o f D IG -lab eled probes in n on-rad ioac tiv e ISH is based on m e thod s deve loped by B ald ino andLew is (1 989 ). W e used a p ro to co l sim ila r to th at used fo r th eA P -con juga ted probes (s ee abov e ) bu t som ew ha t m od ified , main lyin th e de tectio n p ro cedu re . A fter p rehyb rid iza tion fo r 1 h our,p robes (20 -200 ng /m l) w ere d ilu ted in hyb rid iza tion bu ffe r (4 xSSC , 10% dex tran su lfa te , 1 x D enha rd ts so lu tio n [0 .02% po -lyv iny lpyrro l idone , 0 .0 2% F ico ll, 0 .02% bov ine serum a lbum in] ,400 ig /m l fre sh ly den atu red so n ica ted sa lm on sp erm DNA , an d30% de ion ized fo rm am ide ) , and hyb rid ized a t 37# {176 }Cvern igh t(14 -1 8 ho urs ). A f te r h y br id iz at io n , the tissue sectio ns w ere w ashedat a strin gency of ap prox im ate ly 10# {176}Ce low th e m e lting tem -pe ra tu re, Tm (F itzpa tr ick -M cE lligo tt e t a ! , 1 988 ), equ ilib ra ted atroom tem pera tu re w ith 1 x SSC for 10 m inu tes , b u ffe r 1 (0 .1 MT ris -H C 1 , pH 7 .5 , con ta in ing 0 .1 4 M N aC 1) fo r 30 m in u te s, andp re trea ted w ith 0 .3% T riton X - 100 and 2% norm a l sheep se rum(NSS ) in Buffer I fo r 10 m inu tes to reduce nonspec ific b in d ingo f the an ti-D IG A P con ju ga te. F ,b fragm en ts o f an ti-D IG A Pcon juga te w ere d ilu ted 1 :50 0 in B uffe r I, 0 .03% T riton X -100 ,an d 1% N SS and added to the tis sue sec tio ns fo r 3 hou rs . S lide sw ere rin sed and equ ilib rated in 0 .1 M T ris -H C I, pH 9 .5 , 0 .1 MN aC l, and 0 .0 5 M M gC l2 fo r 2 x 5 m inu tes. Fu rthe r, the sl idesw ere in cu ba ted w ith the sub stra te m ix tu re (5 -b rom o-4 -ch lo ro -3 -ind o ly lp hosp ha te /n itro b lue tetrazo lium , [BC IP /N BT ], co n-ta in in g 0 .0 01 M lev am iso l) (Leary e ta ! , 1 98 3). Th e co lo r reactio nw as stop ped by add itio n o f 1 0 mM ethy len ed iam ine tetraace ticacid (EDTA ), an d cov erslip s w ere m oun ted in M oun t-Q u ickAqueou s@.

Rad ioac t ive S P robes-R ad io labe led S o l igodeoxynuc leo -tide p ro bes w ere h ybrid ized to tis sue sec tion s using a p ro toco lbased on the m eth ods d eve lo ped by Y oung e t a ! (198 6). T issu esec tions w ere depara ffin ized an d d igested w ith pro te inase K (20ig /m l) at 37 #{176}Cor 25 m in u tes. A fte r 3 0 secon ds in 0 .2 M glyc ine(in 0 .02 M T ris -H C1, pH 7.5 ), th e sec tions w ere dehyd ra ted anddried . E ach slide w as cov ered w ith 1 07 cpm prob e pe r m l hy-brid izatio n buffer (50% fo rm am id e , 4 x SSC , 10% dex tran su l-fa te , 1 x Denhard ts , 1% sarcosyl , 2 .4 m g/m l N a2H PO4, 40 0 ig /m l sa lm on sp erm DNA , an d 0 .2 M dith io th re ito l [D TT ]) an dhyb rid ized a t 37# {176}Co r 14 -1 8 hours. A fte r th e w ash in g p ro cedu re ,


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3.. .

94 - .6 7 -43- .

30 -

2 0 -14 -

20 J ou rn al o f A n dro lo gy . J an u ar//F eb ru ary 1 99 6

M rX i- Ma b i

*

M ab 25

1 2 3 1 2 3F I G . 1. W este rn b lo ts o f S g l an d S gll u s ing m on oc lo na l an tib od ie s . P artia lly liqu e fied h u m an se m in a l p lasm a (co r res po nd in g to a pp rox im ate ly 0 .05

iI o f se m ina l p lasm a, lan e 1 ), in tac t S g Il (0 .6 , la ne 2 ), an d in tac t S gl (0 .6 g , lan e 3 ) w ere re d uce d an d ru n o n S O S /P A G E . A fte r tran s fe r to a n y lonm e m b r a n e , th e se m e no ge lins w ere v isu a liz ed w ith m on oc lo na l an ti-S g l/S g Il lgG (M ab 1 le ft; M ab 2 5 , r Igh t ) . T he p os itio ns o f m ole cu la r m ass m arke rsa re in dic ate d ( fa r le ft) .

th e sec t ions w ere deh ydra ted , a ir-d r ied , d ipped once in 42 #{176}CIlfo rd K -S au to rad iography emulsion , d ilu ted 1 :1 in D EPC -w a-te r, d r ied fo r 4 hours , an d expo sed fo r 5-14 day s in ligh t-sea ledb oxes at - 20# {176 }C .ectio ns w ere deve lop ed fo r 2 minu te s in D - 19so lu tion , f ixed fo r 3 m in u te s in A geF ix , r in sed in w a te r, cou n-te rs tain ed fo r 45 secon ds in M eyers hem atoxy lin , dehydrated ,and m oun ted in P e rtex . A l! sec tion s w ere ana lyzed u sing anO lym pus-CX m icroscop e w ith b rig h tf ie ld an d da rk field illum i-nat ion .P rocedures to V erify th e Sp ecific ity o f ISH R ea ctio nsA D IG -labe led oh igo -dT pro be serv ed a s the p os itiv e hy brid iz a-tio n co n tro l in a ll tissues. In tissue specim en s con tain ing pro sta teg lands , an A P -co n jug ated or a 35S-labe led an tisense p ro be com -p lem en ta ry to nuc leo tide s 528 -5 57 o f the PSA transc rip t (B jar -te ll e t a !, 199 3) a lso se rv ed as a positiv e h ybrid iza tion co n tro l.

F our d iffe ren t p rocedu re s se rved as nega tive co n tro ls : 1 ) hy -b rid izatio n in the absense o f an y prob e; 2 ) c o m pe ti ti on withincreasing concen tra tio ns of an un labe l ed pro be ; 3 ) hybridizat ionwith an unre lated an tisense p robe , com plem en ta ry to the PSAtranscr ip t (n o t used in p ros ta te tissues) ; an d 4) RNA se p re trea t-m en t o f th e tissu e sec tions (0 .! m g /m l in 2 x SSC , 10 m g M gC l2fo r 1 ho ur a t 37#{176}C)e fo re hy brid izatio n w ith e ithe r th e an tis en sep robe fo r S g I o r tha t com plem en tary to th e SgII transcr ip t.

Re s u l t sC hara cteriza tio n o f M onoclona l S g A ntib od ies andO lig on uc le otid e P ro be sDu rin g the p rod uc tion o f m onoc lon al an tibod ies to SgI,seve ra l po sitive c lones w ere iden tif ied , and a fte r sub clo n -ing , n ine d iffe ren t h ybridom as w ere used fo r the p rod uc -tion of an tibo d ie s . A ll m onoc lon al an tibo d ie s ( i.e ., M abs1, 5, 8 , 9 , 17A , 22 , and 25 ) w ere o f iso ty pe IgG lK . A llclo nes reac ted w ith bo th in tac t S g I and in tac t SgH . W est-ern b lo ts sh ow ed bo th M ab 1 and M ab 25 to reac t w ithin tact S g I a s w ell a s w ith in tac t S g II , b u t also w ith frag -m en ts o f e ithe r (F ig . 1 ) . S om e of the m onoc lona l an ti-b od ie s sta ined Sg I m ore in tense ly than Sg II and v ice ve rsain w este rn b lo ts , b u t the re w ere a lso d ifferences in thereac tiv ity w ith d ifferen t fragm en ts o f S g I an d Sg II .

T o com pare the sensitiv ity o f the d iffe ren t an tibod ies ,a do t b lo t exp erim en t w as p erfo rm ed (T ab le 1 ). Mo s tm ono clo na l an tib od ie s recogn ized 20 tim es h igh er fo r S gII than fo r S gI , andth at o f M ab 1 7A > 80 tim es h ig he r fo r S gI th an fo r S gII.


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B jarte ll e t a l . T is su e E xp re ss io n o f S em en og elin la nd I! 21Tab le 1. D ot b lo t re su lts show ing the sens itiv ity o f m onoc lona lan tibod ie s ra ised aga in s t hum an Sg l

S em en og e lin I S em en o ge lin IIM ab 1


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22 J ou rn al o f A nd ro lo gy J an u ar y/F e bru a ry 1 99 6

F IG . 2 . Im m un oc y toch e m lc a l de m o n s tra tion o f S g l an d S g ll In pa ra ffin s ec tion s o f h um an se m ina l ve s ic les an d ep id id ym is . (A ), M o no c lo n a l a n ti-S g l/S g ll lg G s (M a b 1 ) Im m un o s ta ln c e lls , p re d om in an tly d ee p in the c ryp ts o f the se c re to ry e p ith e liu m . (B ), Lo w -m a gn ifica tion m ic ro g ra p h sh ow in gS g Im mun ore a c tlv ity (M ab 9 ) re s tric ted to the ca ud a l re g ion (a rro w s) In a lo n g itu d in a l sec tio n o f w ho le e p id id ym is . (C ), F re sh ly e ja cu la te d spe rm atoz oaw ere im m un o s ta ine d u s ing a m o n oc lon a l an ti-S g l/S g ll lg G p rep a ra tion (M a b 2 5). A P A A P tec hn iq ue w ith F as t R ed a s a ch rom op h ore s ho w s im m u -n os ta in in g o n the po s tac ros om a l pa rt o f th e spe rm he a d , o n the ta il, a nd the m os t in te n se s ta in in g on the m id p ie ce reg io n . (D ), In accord w it h f in din gs


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ob ta ine d us in g the m on oc lo na l lg G p rep ara tio ns , th e po lyc lona l lg G d e m ons tra ted im mu no re ac tiv ity in the e p ithe lla l ce lls and th e lu m e n co n te n t inth e ca u da reg io n o f e p id idym is . (E ), A dd ition o f the 52 -a m ino -a c id S g l fra gm en t (1 0 pg /m I d ilu ted a n tibo d y prepara t ion , -4 0 x m o la r ex ces s ) p r io r toa p p lica tio n o n the tissu e se c tio ns co m p le te ly p re ve n ts the Im m u n ore a c tio n . B o u in fixa tio n . B a rs : A , B , D , E 1 00 p m ; C 1 0 pm .

B ja rte ll e t a t T is su e E xp re ss io n o f S em e no ge lin la nd !! 23

F IG . 3 . I n s it u hyb r id iza t ion ( ISH ) a n d i m m u n o cy t oc h e m is t ry (IC C) o n p ara ffin sec t ions o f h um an sem in a l ves ic les . (A ), U sin g an A P-co n jug a te d3 0 -m e r a n tise ns e p ro be sp ec ific fo r th e S g l tran sc r ip t, s tron g hyb rid iza tion s ig n a ls w e re d e te c ted In ep ith e lla l ce lls . N ote th e per lnuc lea r sta iningp a tte rn . (B ) , A n a d jace n t se c tio n h yb r id ize d w ith a s im ila r p ro b e spec i f ic fo r S g lI tran sc rip ts , h yb r id ize d to the sa m e ep ith e lia l ce lls , b u t w ith le ssIn te n se s ign a ls . (C ), A n Irre le va n t A P -co n jug a te d p ro be (P S A) g en e ra te s n o h yb rid iz a tion s ign a ls a t a ll. N ote th e In trace llu la r lip o fu sc ln g ra n u les , w hicha re ty p ica l o f ep lth e lia l ce lls o f th e se m in a l ve s ic les . (D ), D ig ox ig en in la be ling o f the S g l p ro b e g en e ra te s a s im ila r s ta in ing h yb rid iza tio n pa tte rn , a ndo n an a d jac en t sec tion (E ), th e s am e ep ith e lla l ce lls Im mu no re ac t w ith the p o lyc lon a l S g -lg G p rep a ra tion . (F ), R a d io la be ling (M S) o f th e S g l-sp ec lflc3 0 -m er a n tise nse p ro be a lso h yb r id ize d to th e sem in a l ve s ic ie ep ith e lum , b u t w ith a m uc h m o re d iffu se pa tte rn . B C IP -N B T s erv ed as th e ch ro m o ph oreIn IS H , an d IC C w a s p erfo rm e d w ith the S A -H R P te ch n iqu e a nd D A B sta in in g . B a rs : A , B , C , an d F = 2 5 pm ; D an d E = 1 00 p m .

4-


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24 J ou rn al o f A n dro lo gy . J an u ary /F e br ua ry 1 99 6

F IG . 4 . N on -ra d ioa c tiv e IS H o f the h um an e p id idym is w ith D IG -lab e le d o lig od eo xyn u c le o tide p ro b es . (B ), T he 30 -m er an tise n se p ro b e sp ec ific fo rthe S gl tra n sc rip t do e s n o t h yb rid iz e to th e e p ithe lia l c e lls , in con tra s t to the w e ll-d e te c tab le hy b rid iza tion s ig na ls o b ta in e d w ith th e S g li sp e c ific p ro be(A) . Ba rs 25 m .

(F ig . 3 ). A p erinuc lea r sta in ing pa tte rn w as de tected in asu bp opu la tion o f ep ith e lia l c e l ls , th e h ig he st dens ity o fs ta in ed ce lls b ein g p re sen t in th e b asa l pa rt o f th e cry p ts .T he sign als w ere co nsisten tly m ore in ten se w ith th e an -tisense p rob e fo r S gI than w ith tha t fo r S gII . T he n on -rad ioactiv e p ro bes cou ld b e used to d em on stra te in de tailtha t ep ith elia l ce lls exp re ssing SgI a lso ex pressed SgII. A P -con juga ted pro bes show ed a be tte r signa l- to -no ise ra tioin the sem ina l v esicle s than d id D IG -labe led pro bes. T heo ligo -dT p rob e lab eled w ith D IG hyb rid iz ed to m o st tis -sue ce lls (bo th ep ith e lia l and s trom al ce lls ) a s a po s itiv econ tro l fo r the techn ique . N ega tive co n tro ls , inc lud inghybrid izatio n w ith irrelevan t an tisense p ro be (PSA ),om ission of p ro be , com pe titio n w ith u n labe led p ro be , an dRNA se d ig es tion of tissue sectio ns p rio r to hyb rid iza tion ,a ll supp orted th e sp ec if icity o f the hy brid iza tio n s igna ls(F ig . 3 ).

T h e S gII-spec ific p rob e gene ra ted hyb rid iza tion sig na lsin ep ithe lial ce lls o f th e cau da l ep id id ym is , w h e reas th eSg l p rob e d id no t (F ig . 4 ). N o hybrid izatio n sign als w ered ete c ted in th e lum ina o f tissu e sec tio ns tha t s till co n -ta ined lum ina l spe rm a tozo a a fte r the p ro ce ss ing o f thes lide s . ISH w ith D IG -labe led p ro be s w as su pe rio r to th atw ith th e A P pro be s b ecause th ey gene rated le ss b ack -g ro un d sta in ing in ep id idym a l t issu e . N one o f th e labe led

an tisense p rob es fo r S g I o r S gII gene rated an y spec if ichy brid izatio n signa ls in the te stis , th e d e fe ren t du cts , o rp ros tate tissue sec tions.

T h in ad jacen t tissue section s o f sem in al v es ic le s an dep id idym is w ere also u sed to com pare s ta ining pat ternso f ISH ob ta ined w ith d iffe ren t p robes, and to eva lua te theco rre la tion of s ta ining g en e ra ted by IC C to stain ing re -actio ns p rod uced by ISH . T hu s, ce lls im m unosta ined fo rSg I/Sg II w e re fou nd to be iden tical w ith th e ce lls ex pres s-ing th e Sg I and SgII tran sc rip ts de tected by ISH (F ig .3D,E).

Di s c u s s i o nThe pu rpose o f the p resen t study us in g IC C and ISH w asto further investig ate th e p ro duc tio n an d d istrib u tion ofSg l an d S gII in d iffe ren t no rm a l h um an tissue specim ens ,pa rticu lar ly in the sem ina l vesic le s an d the ep id id ym is .

T is sues from th e m ale g en ital trac t, ob ta in ed a t su rge ry ,w ere p repa red and fixed unde r con d ition s op tim ized top re serve m RNA fo r ISH stud ie s , a s recen tly desc ribed(B ja r tell e t a l, 199 3 , B jO rk et a l, 199 4). A rch iva l tissuesp ecim ens w e re a lso used fo r IC C becau se the m onoc lon a lan d po lyc lo na l an t i-S g IgG s w o rked w e ll o n ro u tin ely fix ed


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B jarte ll e t a l T iss ue E xp re ss io n o f S em en og e!in la nd II 25tis su e s, fa c ilita tin g an ex ten sive inv es tiga tion o f th e oc -cu rrence o f imm unoreac tiv ity fo r Sg I/Sg II in a m ult ip lic-ity o f o rgans .

M ono clo na l an tibo d ies ag ain s t Sg I o r ag a in s t Sg II h av eno t p rev iou s ly b een av ailab le . N ine d iffe ren t m onoc lon a lan tib od ie s re cogn iz ed bo th SgI and Sg II . I t is like ly tha tth is c ross -react iv ity is d ue to th e ap p rox im ate ly 8 0%struc tu ral s im ila rity b etw een SgI an d Sg II. T he ep itop esw ere no t s tud ied in deta i l , bu t do t b lo t and W este rn b lo texpe rim en ts sugg ested tha t th e an tibod ie s reco gn ized a tle as t fiv e d iffe ren t ep ito pe s tru ctu re s (F ig . 1 ).

In th is study , a ll S g I/Sg II m ono clo na l an tibod ies te stedy ie lded id en tica l re su lts w ith th e p rev iou sly cha rac te rizedpo ly clo na l an ti-S g IgG s. In con tras t to p rev io us s tu d ie s(L ilja e t al, 1 989 ), w e a lso exam ined the d is tribu tion ofS gII w ith in the ep id id ym is and fou nd th e im m unoreac -tiv ity to be con fined to the ep ith elium of the cau da ep i-d idym is, w he re th e spe rm a tozoa a re co llec ted be fo re ejac -u latio n . Th e capu t and co rp us reg ions o f ep id idym is w erecom ple te ly un sta ined . In te re sting ly , th e spe rm a tozoap re sen t in th e lum ina of the tissue spec im ens a lso m an-ife sted Sg imm uno reactiv ity , bu t on ly in th e caud a reg iono f th e ep id id ym is . In con tras t, E vans an d H e rr (1 98 6)w e re un ab le to de te ct any SV SA ep itop es in th e ep id id -ym is w ith th e ir M H S -5 m onoc lon al IgG , bu t it is u nc learfrom th e ir repo rt w he th er th ey exam ined d ifferen t pa rtso f th e ep id id ym is in d eta il. A ll m onoc lo na l IgG s aga ins tSg I /Sg I I d escribed he re a lso im m unosta ined fre sh ly e jac-u la ted spe rm atozoa , a find ing in acco rdan ce w ith th oseof p rev io us stud ie s usin g po ly cio na l an ti-S g I/S g II IgG s(L ilja e t a l , 19 89 ), and w ith fin d in gs rega rd in g the SV SAan tig en a s stud ied b y H err e t a l (1 98 6).

B ecau se ne ith er th e p o ly clon al an tib od ies ag ain s t Sg I/S g II no r the m onoc lon a l an ti-S g IJSg II IgG s d esc ribed he reare spec ific fo r e ithe r S gI o r Sg II, w e u sed ISH to dem -onstra te sp ec if ic tran sc rip ts in tissu e sec tion s. It p ro vedposs ib le to d esig n o ligod eo xynuc leo tide p robes spec ific tothe re spec tive m RNA fo r S g I and SgI I , and th e con stru ct-ed p robes show ed c ross-hy brid iza tion to be < 0 .5% . T henon -rad io ac tiv e ISH m ethod s w e re fou nd to b e su pe rio rto th e rad ioact ive p ro cedu re in tha t they d is tin ct ly v isu -a liz e th e pe rinu c lea r s ta in ing pa tte rn in ce lls ex pre ss ingthe spec ific transcr ip t, in con tra st to ad jacen t un sta inedce lls (F ig s. 3 , 4 ) . U sing rad ioac tive p robes and au to ra -d iog rap hy it w as m o re d ifficu lt to de te rm ine exactly w h ichce lls ex pre ss a sp ec if ic m RNA (F ig . 3F) .

A bund an t ex p re ssion o f S gI an d S gII in the sem ina lv es icle ep ithe lium has p rev ious ly b een dem on stra ted b yN orthe rn b lo t stud ie s , cD NA clo n ing ex pe rim en ts , an dIC C (L ilja e t a l, 19 89 ; L ilja and L undw all, 19 92 ). U singISH , th e SgI and SgI I transcr ip ts w ere s h own h ere to b eh igh ly exp ressed in th e sem in a l ve s ic les , w ith a typ ica lp erinuc le ar d is tribu t ion pa tte rn o f m RNA . U sing ad ja -c en t se c tion s , the Sg I and SgII transc rip ts w ere fou nd to

be expre ssed in the s ame ep ithe lia l ce lls . In the ep id idy -m is , w e iden tified h yb rid iz a tion s ig na ls sp ec ific to the Sg IItran scrip t, w he rea s th e Sg I transcrip t co u ld no t be d e-tec ted . T h e exp ress ion o f S gII w as co n fin ed to th e ep i-the lium o f the caud a reg io n . N o hyb rid iz a tio n sign als fo rth e SgI o r SgII tran scrip ts w ere de te cted o n spe rm ato zo ap resen t in the lum in a o f the capu t, the co rp us, o r caudaep id id ym is o r te s tis , ind ica tin g tha t im munoreac tiv e SgIJSg II o n sp erm atozoa m ay rep re sen t a ttach ed p ro te in s d e -riv ing from th e cau da ep id id ym is sec re tio n . W hethe r itrea lly rep re sen ts S g II fou nd on the sp erm a tozoa needs tobe c la rif ied . W e w ere u nab le to id en tify any sign als o f S gIm RNA p resen t in the ep id id ym is , S g I and Sg II m RNAin the de fe ren t du cts , p ros tate , o r tes tis , a re su lt tha t fu r-the r supp orts b o th p rev io us and p re sen t im m unocy to -chem ica l find ing s .

S gI an d SgI I have been sh ow n to b e re spon sib le fo r th eim med ia te ge l fo rm ation of fresh ly ejacu la ted sem en (L iii aan d L aurell, 1 984 , 198 5). T he func tion of Sg II p rodu cedin th e cauda reg ion o f the ep id idym is and th e p resen ce o fsem enoge lin ep ito pe s o n the sp erm a tozoa rem a in to beestab lish ed . T hese fin d ing s m ay b e im po rtan t fo r an un -d ers tan d in g o f n o rm a l g el fo rm ation and no rm a l sp ermfu nc tion , a ltho ug h Sg I an d S gII m ay a lso have o th er fun c -tio ns. In th is investig atio n the find in gs o fim m unoreac tiveSgI/S gII in a m ultip licity o f h um an tissu es ve rified th attissu e ex pre ss ion of sem enog elin s is p redom inan tly co n-fin ed to th e sem in al vesic le s an d the cau da ep id idym is,and thu s the an tib od ie s m ay b e used as spec ific marke rsof sem in al vesic le tis sue in co re b iop sie s from the prosta teg lan d or fo r cauda ep id idym is spec im ens.

aflW flI S W V W IflIW I 5

W e thank E lise N ilsson an d V inka F ilin ic a t the D epartm en t o f P atho l-o gy , B ir gi tt a Frohm an d In gr id W ig he de n and S anna Hu lk ko at theDepar tmen t o f C li ni ca l Chemis t ry , Lund U nivers ity , U n iversity H os-pital, Malm#{246 } ,w eden , fo r th eir exper t t ec hn ic al a ss is ta nc e.

Re f e r e n c e sA k i y a m a K , N akam ura T , Iw an aga S , H ara M . The ch ym otryps in -like

activity of the pros ta te -spec ific an tigen g am ma-sem ino p ro te in . F E B SLett , 1987;225:168--172 .

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Distribucion y Expresion Tisular de La Semelogelinas

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