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Su ppo rte d b y a g ran t from T he N ationa l H ealth and M edicalR e sea rch Coun cil o f A us tra l ia and the Q u een slan d Cance r Fund .

677

0022-1 554/82IO675SO2.5The Journal o f H isto ch em istry and Cy tochem istryC opyrig h t 1982 by Th e H is tochem ic al S ociety , Inc .

Vo l . 30 , No . , pp . 6#{ 149}-#{244}81 . 1982P ri nt ed i n U .S .A .

IINuc lea r D iam eter in th e A n ter ior P itu itary G land o fth e R a t:E ffects o f E s trog en , B rom ocrip tin e , and H alop erid oPJ .M . JA COB I, H .M . LLOYD , and J .D . M EARESD epa rtm en t o f M ed ic in e . U n izer sity o f Q ueensland . C lin ic a l Scienc es B u ild in g . Ro ya l B risb an e H o sp ita l. B risban e 4 02 9 . Au stra lia

Received for p ublicat ion F ebruary 9 , 1 981 and in rev ised fo rm D ecem ber 2 2 , 198 1 ; a ccep ted D ecem ber 3 1 , 198 1 (O A 81-126 )

N uclear d iam eter , D N A syn thesis, and m ito tic ind ex inthe p itu itary ce lls o f m ale ra ts and serum pro lac tin w erem easu red after a period of 8 days o f trea tm en t w ith ado pam in e agon is t and an an tag on ist g iven w ith and w ith -o u t estrog en . In the absence of es trogen , the d opam ineagon is t, b rom ocrip tine , d im in ished th e m ean nu c lea r d i-am eter o f the p itu ita ry ce lls and low ered p itu ita ry D NAsyn thesis , and the dop am ine-b lo ck ing ag en t ha lop en id o lhad no effect. Es trogen increased the m ean nuc lea r d i-am eter , p itu ita ry m ito tic ind ex , and D NA syn thesis . B rom -ocrip tine preven ted th e es trogen-ind uced increase in m ean

Introduc t ionE arly stud ies o f endo crine tissue revea led th at nu clea r h ype r-tro ph y w as a sso cia ted w ith in cre ased se cre to ry a ctiv ity andw ith grow th (4) . M ore recen tly , nu clea r s ize (d iam e ter) ofpa rathyro id aden om a ce lls w as fo und to b e re la ted to p lo id ity(3 ) and to be correla ted w ith tum or w e igh t ( 16). In a stu dyof parathy ro id ad enom as no co rre la tion w as fou nd b etw eenm ean nu cle ar d iam e ter of tum or cell nu cle i a nd pa rath yro idDN A synthes is, a lthough 76% of tritia ted thym id ine-labe led(S phase) n uc lei m easu red 6 j. tm (1 7). In the ca se o f the p i-tu itary g land , li ttle in form a tion ab ou t nuc lea r siz e is av aila ble .C h an ges in shape an d den sity o f nuc lei w ere observed in p i-tu i tary ce lls taken from cas tra ted fem ale ra ts (8 ) and in p itu ita rychrom ophob e ce lls o f ra ts g iven cor tiso l (12). S tud ies o f thepitu ita ry g land in w hich var iou s ag en ts in fluence b oth D NAsynth esis and secre tio n h ave pro v id ed oppo rtun itie s to inv es-tig ate th eir re la tionsh ip w ith nuc lea r d iam e ter. F or exam ple,estro gen affec ts p itu ita ry ce ll d iv is ion and secre tion of p ro -lac tin (13 ). A lthou gh ce ll hyp ertrop hy w as o bse rved in ratp i tu itary cel ls trea ted w ith estrad io l , nuc lea r size w a s sta ted

nuc lear d iam ete r an d p itu itary D N A syn thes is an d m ito ticin dex w ere low ered . H aloperido l augm ented the estro gen-in duced increase in m ean nu clea r d iam ete r, p itu ita ry D NAsyn thesis , and m ito tic index . Po sitiv e co rre lations w ereob ta ined b etw een m ean nu clea r d iam eter and D NA syn -thesis an d serum pro lac tin . It w as conc luded th at nuc leard iam ete r w as in fluenced by bo th D NA syn thesis and se-c re to ry ac tiv ity in p itu itary ce lls.KEY WORDS . P itu ita ry ; N uclear d iam e ter ; DNA syn the sis;B rom ocrip rine; H a lop erid o l.

to be s im ila r to tha t o f un trea ted cells ( 1 ). The nuc lea r sizeo f p ro lac tin ce lls ob ta ined from ra ts trea ted w ith brom ocn ip -tin e app ea red to be red uced (9). B rom ocrip tine is a dop am ineagon ist kn ow n to inh ib it p ro lac tin sec re tio n and to suppresse stro gen -in duced m ito tic act iv ity in the an te rio r p itu itary ofthe ra t (13 ,18). T here is n o in fo rm ation w ith regard to th eeffec ts on nuc lea r d iam ete r o f dopam ine an tag on ists (excep tes tro gen) tha t inc rease the sec re tion of p ro lactin . W e haveprev io usly show n th at ha loperido l g iven by itse lf inc reasesp ro lac tin sec re tio n and stim ula tes bo th pro lac tin secretion andp itu i tary D NA synthes is w h en given w ith es tro gen (15) . Th epu rpose of the p resen t inves tiga tion w as to exam ine the effec tso f these ag en ts on n uc lea r d iam ete r and to de te rm ine w hethernu cle ar siz e is sp ecifica lly rela ted to DNA synth esis o n theon e hand or to secreto ry ac tiv ity o n the o ther.

M ateria ls an d M etho dsM aterials . D ie thy ls tilbes tro l d ip rop ion a te (D ESD ) (M ay and

B aker) w as g iven in a veh ic le o fe thy l o lea te a t a con cen tra tion of 10m g pe r m l. B rom oc rip t ine (S andoz, B a sIc) w as d isso lved in a v eh iclecon tain ing an equ al w e igh t o f p o ta ssium hyd rog en tartra te , 1 p arta bso lu te alcoh ol, a nd 6 p arts sa line . H a lop erid o l (Se arle , A ustra lia)w as d isso lved in a v eh icle co nsis ting of 0 . 1 lac tic a cid in sal ine .[H J-Thym id in e (spec ific act iv i ty 1 5-3 0 C i/mm ol) w a s purcha sed fromthe R adio chem ical C entre , Am ersham .

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67 8 JA CO B I, LLO YD , M EARES

M N D wa s m easu red in app ro x im ate ly 30 to 6 0 nuc lei rad ioau to graph ical ly lab ele d .hN ot m easured . Te st too insens itiv e to dem on stra te m ino r ch ang es from no rm al.

A n im als . M ale Sprag ue-D aw ley ra ts , aged 3 m on th s, w ere kep tin a lig h t- a nd tem peratu re -co n tro lle d environm ent and m ain ta inedo n tap w ate r an d a pe lle t d ie t ad l ib i tum . G ro ups o f sev en ra ts ea chw ere g iven a d aily in ject ion of brom ocnip tine (0 .3 m g/tOO g bodyw eig h t) or ha lop en ido l (0 . 1 5 m g/l00 g body w e igh t) or th e veh icle at04 :00 hr for a pe rio d of8 days. A ll the ra ts w e re k illed o n th e m orn ingof th e eig h th day by se ction o f the neck ar ter ies du ring ch lo rofo rman esth esia . P i tu itary g land s w ere rem ov ed an d ha lved sag itta lly . B loo dwa s k ep t fo r ra dio im m un oa ss ay .

R ad io imm unoassay of p ro lac tin . R ad io imm unoassay of p ro lac -tin w as car ried out using N IAMDD rat p ro lact in RP -l a s re ferencepreparatio n a cco rd ing to m ethod s described prev ious ly (1 0).

Pitu ita ry D NA syn thes is and rad ioau tograph y . P itu ita ry D N Asyn the sis w a s est im ated (in v itro ) in o ne-half o f ea ch pitu i tary g landas describ ed prev ious ly (1 0). The ha lf p itu itary g land w as in cub atedfor 3 hr in m ed ium 199 contain ing 1 pC i/m I [3H } -thym id ine ; th eDN A w as ex tracted by Bu rtons m ethod (5 ) and the rad ioac tiv ity inth e D NA w as co un ted . T he o ther ha lf p itu ita ry g land , incuba ted asfo r th e estim ation o f DN A syn thes is , w as fixed in 1 0% form alin insaline and em bedd ed in p ara ffin . Sec tio ns o f4 p .m th ick nes s w ere cu tand p roc essed th rou gh xylo l, abso lu te alc oho l, an d 70 an d 50% alcoh olto w ate r. T he slides w ere coa ted w ith p ho tograp h ic em ulsio n K 2B(Ilfo rd ) h ea ted to 4 5#{176}Cn a w ate r ba th . A f te r exposure in to ta l da rk nessfo r 5 d ays , the sect ion s w ere d eve lop ed an d s tain ed w ith 0 . 1 % Nucle arF ast R ed in 5% alum in ium su lfa te, fo llow ed by a w ash in 0 . 1% picr icacid in abso lu te alc oho l. Th e num ber of lab ele d ce lls w a s cou nted andex pressed as lab e l index p er 1000 cells. Th e to tal num ber of c ellscou nted for ea ch gro up rang ed from 80,000 to 160 ,00 0 .

N uclea r d iam ete r. S ections o f p itu ita ry tis sue (4 m th ick ) , p re -p ared as describ ed abov e, w ere hy dra ted , rinsed in co ld N HC 1, andhyd ro lyzed in 5 N HC I fo r 2 hr a t 2 0#{176}C .f te r one rinse in co ld NH C I and severa l rinses in d is tilled w ate r, the slides w ere s ta ined w ithS ch ifrs reag en t (d e Tom asi m ethod ) for 1 hr, rin sed in a so lu t ion of0 .5% potassium m e tab isu lfite in 5% N HCI, then in w ater , beforebe ing dehyd rated and m ounted in Polym oun t (1 9). M ean Nucle arD iam ete r (M N D ) w as estim ated o n th e Feu lgen-sta ined sec tio ns w iththe a id of a W ild ocu la r g ra ticu le , as descr ib ed prev iously (16) . A p-p rox im ate ly 10 00 nu clei w ere coun ted in e ach sect ion . M ND w as alsoe stim ated on the (5H ]-thym id ine- lab ele d nu cle i sta ined w ith N uc lea r

Fas t R ed , as d esc ribed above . It sho u ld be n o ted tha t the th in ner thesec tion s and the larger the n ucle i, the gre ate r w ill b e the n um ber o fapp aren tly sm all nuc lei in a cu t sect ion . S om e skew ness in d is trib u t ionof nu clea r d iam eter m ay re su lt from the u se o f 4 m sec tio ns in th osep itu ita ry g lan ds w ith la rge nu cle i.

C ytopho tom etry m easurem en ts o f n uc lea r a rea. The Feulg en-s ta in ed s lid es w ere exam ined under a V ickers M 86 scan n ing m icro -d en sitom e ter w ith a 1 .3 N A x 100 apo chrom atic ob jective , a 4 0 md iam eter f ie ld , i llum in ated , a nd a fly in g spo t o f 0 .48 tm and 0 .95tm diam eter . A bou t 100 nuclei f rom each se ction w e re exam in ed .A bso rb anc e an d are a m easurem ents o f the n ucleus w ere no ted .

M ito tic ind ex . Fu rth er group s of rats g iv en v ehicle , o r DE SDw ith an d w ith out brom ocr ip tine or h alo per ido l, as described ab ove ,w ere g iven co lch ic in e (0 . 1 m g p er 1 00 g body w eig h t) in traperitonea lly90 mm before the rats w e re k ille d . W ho le p itu ita ry g lan ds w e re fixedand sta ined w ith h em ato xy lin and eo sin an d the ce lls in m itos is w erecou nted (13 ).

Sta tistic s. S ign ifican ce of d iffe rence w as ca lcu lated b y S tuden tst te s t. C orrela tion coeffic ien ts (r) were c alc u la ted for log v alu es o f th evar iab les.

Resul tsThe resu lts fo r a ll va riab les (m ean SEM ) fo r the s ix g roupsof rats g iven brom ocrip tine , ha lopen ido l, o r veh ic le w ith orw ith ou t estrogen are show n in T ab le I . F ig ure 1 is com po sedof h istog ram s of the range of nuc lea r d iam ete rs (4 .5 26-7 .320p .m ) e stim ated f rom F eulgen -sta ined sections . T he sm allestm ean nuc lea r d iam eter (M N D = 4 .526 , tam ) w as o bse rved inbrom ocnip tin e-trea ted ra ts and th e m ean value w a s low e r thanth at o fthe co n tro lg rou p (p

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50

050 b

5g

Tab le 2 . Number o f nud ei m easu ring 3 to 1 0 p m ind iam e te r exp re ssed as % of th e to ta l nu c lei cou n ted for eachtr ea tm en t g rou p

5-0ci cizdz

e

fr -4 T

5g

0

NUCLEAR D IAM ETER IN THE RAT P ITU ITARY GLAND 67 9

0

0C

U, QI 5#{176} d

050

rT I1 L, ,um 2 34 5678910

F igu re 1 . H is tog ram s repre sen tin g the d istrib u tio n of n uclear d iam -ete r (gm ) in ap pro x im ately 1 000 pitu ita ry c ells from gro ups of ra tsg iven a d opam ine ag onist and an an tago nis t w ith and w itho ut es trog enfor 8 days a s in d ic ate d b elow . (V alu es no ted a re m ean an d SEM ,re spe ctively .) (a) B rom ocr ip t ine (0 .3 m g/tOO g body w eig h t) M ND= 4 .526 0 .03 4; (b) D ESD ( 10 m g) + brom ocn ip t ine M ND =5 .24 7 0 .2 43 ; (c ) ha lop erido l (0 . 1 5 m g/1 00 g body w eig h t) . M ND= 5 .346 0 .188 ; (d) v eh icle M ND = 5 .609 0 .200 ; (e )D ESD MN D = 6 .616 0 .216 ; (1 ) DE SD + halope rid o l M ND= 7 .320 0 .132 . The co lum n show ing nuc lei m easuring 7 jim ha sb een shad ed to d em ons tra te th e in crea se in MND .

crease in M N D . In th e group g iven ha loperido l alon e, theM N D w as no t d if feren t from tha t o f con tro ls nor from tha tof the group giv en estrog en w ith brom ocn ip t ine. T ab le 2 g ive sdata o n th e num ber of nu cle i m easu ring 3- 1 0 tm in d iam e ter,exp res sed a s a per cen t o f th e to tal nu cle i cou nted , fo r e ach

D iam e ten in zmG roup 3 4 5 6 7 8 9 10

B rom ocrip tine 5 44 40 9 1 0 0 0H alo pen ido l 1 14 45 32 7 3 0 0V ehicle 2 17 26 30 16 3 0 0E strogen 0 5 11 28 35 20 4 0Estrogen + 4 19 43 25 5 3 0 0

bromocn ip t ineEs trogen + 0 0 0 1 8 34 30 11 2

halopen ido l

g roup of rats . M ND of tr itia ted thym id ine-labe led nuc le i o fthe rad ioau tograph s m easured from 5.284 tm (in the g roupg iv en ha lo pen ido l) to 6 .380 .am (in th e g roup g iv en estrog enan d ha lop erid o l) (T ab le 1 ). The M N D s of labe led n uc le i w eresim ila r to th ose o f un labe led n uclei, an d o nly th ose o f theg rou ps g iv en b rom ocnip tin e and e strogen w ith h alo pen ido lshow ed sign ifican t d iffe ren ces (P

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6 ..

n s

8

7

6

05z

I. L* ns ns ns *a b C d e f

F igu re 2 . M ean nuclear d iam e ter (M ND ) of a ll n uc lei (b lack circ le s)and of tr itia ted -la bele d n uclei (open circ les) fo r each g rou p . (a ) brom -ocn ip tine; (b ) es tro gen + brom ocn ip tine; (c) halope rid o l; (d ) veh ic le ;(e) estrog en; (I ) estrog en + ha lop erido l . S tud en ts t test b etw eenlabeled an d unlabe led M ND fo r e ach gro up , #{176}

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NUCLEAR DIAM ETER IN THE RAT PITU ITARY GLAND 68 1

be ex p la in ed by a phase no t fou nd w ith ha lopen ido l o n ly ,po ssib ly late G 1 . T he larges t M ND found w ith ha lop en ido land es trogen m ay be exp la ined by an increase of ce lls in lateG i or p re -S phase , s ince th is com b in a tion no tab ly stim ula tesD NA syn th es is. In the o ther d irection , the presence o f nuc le ireduced in size (b rom ocrip tine ) is assoc ia ted w ith a s ign ifican treduc tion in D NA syn thesis , bu t S -phase nuc le i w ere o f no r-m al, no t red uced , d iam e ter . T hu s, b rom ocnip tin e, w hile a f-fe ctin g a h igh pro por tion of p i tu itary c ells (low e rin g M ND ),sp are s a gro up of c ells th at rem ains ab le to en ter S pha se w itha no rm al n uc lea r d iam ete r.

O ur absorbance m easurem en ts o f F eu lgen-sta ined nuc le ial low ed u s to draw no conc lus ion s on pitu itary DNA con ten t.O n the o ther hand , the re la tio n of nuc lea r d iam ete r to p lo id ity( 3 ) and th e la rge siz e o f nuc lei s een in the p resen t ex per im ents ,sugges ts tha t po lyp lo idy m ay b e presen t a t th is ear ly stage o festro gen ac tion . Po lyp lo idy has been observed in p itu ita rytum ors induced by es trogen in the ra t (23), in h um an p itu ita rytum ors (7 ), an d in G H 3 cells iso la ted from rat p i tu itary tum ors(6 ) .

N o te added in pro of. S ince th is paper w as su bm itted oura tten tio n has b een draw n to the artic le b y Poo le M C , M ah eshV B , K os to ff A (A m J A nat 158 :3 , 1980 ) in w hich nuc lea rv o lum e in ra t p itu ita ry m amm otrop es w as m easured du ringthe es trus cyc le , show ing in particu la r a rise in nuc lear v o lum ew ith rising estrad io l le ve ls.

L ite ra tu re C ited1. An tak ly T , P elle tier G , Z ey tino g lu F , Labrie F : C hange s of c ell

m orp hology and pro lac tin se cre tio n indu ced by 2 -B r-e rgo cn ip -tin e , estrad io l, a nd th yro tro p in -releas ing ho rm one in rat an terio rpituitary ce lls in cu ltu re . J C e ll B io l 86 :3 77 , 19 80

2. A ttram ad al A : C ellu la r loc aliz ation of H -o estrad ioI in th e b y-pophysis. Z Ze llfo rsch M ikrosk Anat 104 :597, 1970

3. Bengtsso n A , G rim elius L ,Jo han sso n H , PontenJ: N uc lea r D NA -conten t ofp ara thy ro id ce lls in adenom as , hy pe rp la stic and norm a lg lan ds. A cta Patho l M icro b io l S cand 85A :45 5 , 1 977

4. B rache tj, M insky A E , E ds: T he C ell, v o l 5 . A cadem ic Press, N ewYo rk , 1 961 , p 49-64 , 4 13-4 14

5. Burton K : A stud y of the cond itions and m echan ism of the d i-ph en ylam in e rea ction for co lor im e tric est im a tio n of d eo xyn ibo -nuc leic acid . B io chem J 62:3 15 , 1 956

6. C lausen O PF, G au tv ik K M , L indm o T : C ell cy cle d is tribu tions ,g row th ch ara cte rist ics, an d va ria t ion in pro lac tin an d grow th h or-m one pro du ctio n in cu ltu red rat p i tu i tary c ells . V irchow s A rchCell P a tho l 2 3 :195 , 197 7

7. F and SB : P o lyp lo idy in the hum an p itu ita ry : co ex istence o f h y-p erp lasia and hypertrop hy . Am J P ath ol 70 :61 a, 1 973

8. F arquhar M G , R inehart JF : E lec tron m icroscop ic stud ies o f thean ter io r p itu i tary g lan d of cas tra te rats . E ndo crino logy 54 :51 6 ,1954

9 . G rafK J, H orow sk i R , E l E treby M F: E ffec t o fp ro lactin inh ib ito ryagen ts on the ec to p ic an ter io r p itu ita ry and the m amm ary g landin rats . A cta Endocn ino l 85 :2 67 , 1 977

10 . Ja cob i JM , L loy d HM , M ea res JD : O n se t o f o estrog en- ind ucedpro lac tin secretio n an d DNA syn th esis by the ra t p i tu itary g land .J Endo cninol 72:35, 197 7

1 1 . Ja cob i JM , L loy d HM : M odu lation b y dopam in e an tag onists o fDN A synthes is in the p itu ita ry g land of th e m ale ra t. N euroen-do cn ino log y 33:9 7 , 19 81

12 . Kraicer J , H erlan t M , D uclos P : C hang es in adenohypophysea lcy to logy an d nu cleic ac id co nten t in th e rat 3 2 d ays af ter b ila tera ladrena lec tom y and the ch ron ic in jec tion ofcortiso l. C anJ P hy sio lPh a rmaco l 45:947, 1967

13 . L loy d HM , M eare sJD ,Ja cob iJM : Ea rly ef fec ts ofstilboe stro l o ng row th horm one an d p ro lactin sec re tio n and on p itu ita ry m ito ticact iv ity in the m ale ra t. J E ndocn ino l 5 8 :227 , 1973

14 . L lo yd HM , M ea resJD ,Jaco biJM : E ffec ts ofo estrog en an d b rom -ocr ip t ine on in v ivo se cre tion and m itos is in p ro lact in cells. N atu re(Lond) 255:497, 1975

15 . L loy d HM ,Jaco biJM , M ea resJD : D NA synth esis and d epletio nof pro lac tin in the p itu ita ry g land of the m ale ra t. J E ndo crin o l7 7 :129 , 1978

16 . L lo yd HM ,Ja cob iJM , C ooke RA : N uc lea r d iam eter in parath y-ro id ade nom as . J C lin Pa tho l 32 :1278 , 1979

1 7. L lo yd HM , Jaco bi JM , W illgo ss D , K earney J , W ard P : D N Asyn the sis and se cre to ry a ctiv ity in parath yro id adenom as . A c taEndoc rin o l 96 :70 , 19 81

18 . P aw likow sky M , K unert-R adek J: E ffec ts o f estrad io l an d apo -m orph in e on the an te rio r p itu ita ry ce ll p ro life ra tion in ov an iec -tom ized fem a le rats. N euro endocr ino l Let t 4 :22 9 , 19 80

19 . P earse A GE: H istochem istry .J & A Church ill L td ., L ondon , 1 96820 . S tep ien H , W olan iu k A , P aw likow sk i M : E ffec ts o fp im oz id e an d

b rom ocnip tin e on ante rio r p itu itary ce ll p ro lifera tion . J N euralTransmi ss 42:239, 1 978

21 . Stra tm ann IE , E zn in C , Se lle rs EA : E strogen -in duced transfo r-ma tio n ofsom ato tro ph s in to m ammo tro phs in th e ra t. C ell T issu eRes 152:229, 1974

22 . Su rks M I, D efes i CR : D ete rm ina tion of th e ce ll num ber o f eachcell type in th e an te rio r p itu ita ry of eu th yro id and hypo thy ro idrats . E ndocrino lo gy 101 :9 46 , 1 977

23 . W aelbroeck-V an G ayer C : T um eurs hy pophy sa ires ind u ites parles oestro genes chez le rat. II E tude cy togen etique . E urJ C ancer5:11 9, 1969