Irena DZIERŻY KRAY-ROGALSKA* The Connection Between the...

P O L S K A A K A D E M I A N A U K — Z A K Ł A D B A D A N I A S S A K Ó W

A C T A T H E R I O L O G I C A

Irena D Z I E R Ż Y K R A Y - R O G A L S K A *

The Connection Betw een the Endocrine Activity of the Parotid Salivary Glands and Parathyroid

on the Basis of Morphological Pictures

Powiązanie czynności (lokrewnej ślinianek przyusznych i przytarczycy na podstawie obrazów morfologicznych

[With 32 Figs. & 4 Tables]I. In tro d u c tio n ........................................................................................................................ 216

II . R ev iew of l i t e r a t u r e ...............................................................................................................216II I . M a te ria l, m e th o d s and t e c h n i q u e .................................................................................. 222IV . E ffec t of th e re m o v a l of th e p a ra th y ro id g lands on th e m orpho log ica l

p ic tu re of th e s a liv a ry g lan d s of th e w h ite r a t .........................................................2231. O p era tio n t e c h n i q u e ............................................................................ 2232. D esc rip tio n of m a t e r i a l ...................................................................................................... 2243. R e s u l t s ....................................................................................................................................226

V. E ffec t of re m o v a l of th e p a ro tid sa liv a ry g lands on th e m orpho log icalp ic tu re of th e p a ra th y ro id g l a n d s ................................................................................... 2261. O p era tio n t e c h n i q u e ...................................................................................................... 2262. D escrip tio n of m a t e r i a l ...................................................................................................... 2273. R e s u l t s ................................................................................................ 229

VI. E ffec t of a d m in is te r in g p a ro t in on th e m orpho log ica l p ic tu re of th e p a ra th y ro id g lan d s and th e p a ro tid sa liv a ry g l a n d s ................................................230A. P a ra th y ro id g lan d s

1. D esc rip tio n of m a t e r i a l .............................................................................................2312. R e s u l t s .......................................................................................................................... 23i

B. P a ro tid s a liv a ry g lan d s1. D esc rip tio n of m a te r ia l ................................................................................... 2312. R e s u l t s ..........................................................................................................................232

V II. E ffec t of re m o v a l of th e sa liv a ry g lands and a d m in is te rin g p a ro tin onth e au to ra d io g ra p h ic p ic tu re of th e bones of w h ite r a ts . . . . 2321. D escrip tion of m a t e r i a l .............................................................................................2332. R e s u l t s ....................................................................................................................................234

V III. S ta tis tic a l an a ly s is of m a te r ia l . . . . . . . . . 234IX . C onsidera tion of re s u lts an d d is c u s s io n .........................................................................242X. S u m m a r y .................................................................................................................................. 247

R e f e r e n c e s ................................................ ............................................................................ 248S treszczen ie .......................................................................................................................... 255

* P re s e n t add ress: A cadem y of P hysica l E duca tion , D ept, of B iology, W arsaw 45, M arym oncka 90,

1215]

216 I. D zierzykray-R ogalska

I. IN TRO D U C TIO N

R ep o rts h av e ap p ea red in li te ra tu re fo r som e considerab le tim e on th e su b je c t of th e p o ss ib ilitie s of th e endocrine ac tiv ity of the sa liv a ry g lands ( B u d g e s , 1928)

D u rin g th e p e rio d from 1929—1944 Jap an ese re sea rch w o rk e rs ( O g a t a , 1953: I t o e t al., 1955; 1956) published a la rg e n u m b er of s tu d ies based on physio logical, b iochem ical an d h istochem ical m ethods, in w hich they s ta te d th a t th e sa liv a ry g lands, in a d d itio n to th e ir secre to ry ac tiv ity (digestive), a re also en docrine g lands. In ad d itio n , in 1944 th ey ob tained from sa liv a ry g lands a bio logically ac tiv e su b s tan ce of a p ro te in o u s n a tu re , affecting th e ca lc ification of h a rd tissues and cap ab le of red u c in g th e le v e l of calcium in the blood. This substance , considered to be the specific h o rm o n e of th e sa liv a ry g lands, w as te rm ed by th em p a ro tin .

As is w ell k n o w n , th e p a ra th y ro id g lands a re th e ductless g lan d s specially re sp o n sib le fo r th e phosphorus and calc ium supply to th e organ ism . I h av e not, how ever, e n co u n te red in lite ra tu re , stu d ies of th e in te rd ep en d en ce of th e se tw o g lan d s (sa liv a ry g lands and p a ra th y ro id glands), th e ac tiv ity of w h ich is connected w ith th e leve l of calc ium in th e blood serum .

II. REVIEW OF L ITER A TU R E

In 1909 P a r h o n and G o l d s t e i n devoted considerab le space in th e ir h a n d book on endocrino logy to the group described by M i k u l i c z (1892), ch a rac te rised by th e sy m m e tric a l h y pertrophy of th e sa liv a ry and la ch ry m a l g lands. T hey s ta te d th a t th is d isease is connected w ith th e thy ro id g land (P a r h o n e t al., 1957).

P a r h o n & K a g a n (1923), in th e ir investiga tions of th e p a ro tid sa liv a ry g lands, show ed th a t th e nucle i of the cells lin in g th e lum en of th e d isch a rg in g d u c ts a re s i tu a te d in th e ap ica l p a rt of th e cell, w h ile they found accu m u la tin g sec re tio n in th e basic p a r ts of th e cells ( P a r h o n e t al., 1957). T hese observa tions m ad e i t po ssib le to re a c h th e conclusion th a t the secre tio n of these sa liv a ry g lands is d ischarged not, as is u su a lly th e case, into th e lum en of th e duct, b u t in to th e su rro u n d in g co n n ec tiv e tissu e or to th e vessels.

S ince th a t tim e num erous studies have been pub lished by th e R u m an ian school of P a r h o n on th e sa liv a ry glands and th e ir connection w ith o th e r d u c tle ss g lands ( P a r h o n e t al., 1955— 1957).

S ince 1928 th e p rob lem of th e sa liv a ry glands has occupied th e a tte n tio n of a la rg e n u m b e r of J a p a n e se research w o rk ers . O g a t a endeavoured to find th e an sw er to th e q u es tio n as to w h e th e r ductless ac tiv ity of the sa liv a ry g lands ex is ts . In th e f ir s t p lace he su sp ec ted th a t the sa liv a ry g lands p lay a ce rta in p a r t in d iab e tes and connected th e ir secre tion w ith ca rb o h y d ra te m etabolism .

In connection w ith th e above observations, experim en ts w ere in itia te d on an im als and since th a t tim e num erous re p o r ts have appeared in w orld l i te r a tu re on th e s u b je c t of th e in te rd ep en d en ce be tw een th e sa liv ary g lands and th e end o crin e g lands (G r a n d o s, 1951; H a r v e y , 1952; R a y n a u d , 1954 a, b; A b o u h a r b , 1955; B i x t e r e t al., 1957, 1959; A t k i n s o n , 1959; S h a f e r e t al., 1960; S o s z k a & K r a w c z u k , 1960 a, b; K a w a d a, 1962).

L a c a s s a g n e (1940) was th e f i r s t to find sex d im orph ism of su b m a x illa ry sa liv a ry g lands in m ice. The sa liv a ry g land of the m ale ch a rac te rised by th e p r e d o m in a tin g n u m b e r of g ran u la r tu b es , w h ile the sa livary g lan d of th e fem ale has a m o re v e s icu la r s tru c tu re . (The su b m an d ib u la r sa livary g land in ro d en ts consists of serous vesicles an d tubes, in w hich th e presence of g ra n u la r m a tte r has been co n firm ed and w h ich w ere fo rm erly te rm ed m ucous vesicles. As h is tochem ical m e th o d s

P aro t id sa l iv a ry g lands an d p a ra th y ro id g lands 217

hav e no t d em o n s tra ted th e p resen ce in th e se e lem en ts of m ucus , th ey a re now te rm ed g ra n u la r tubes). T h is au th o r in je c te d fem ale m ice w ith m ale sex h o rm o n es (te s to ste rone) and m ale m ice — w ith fem a le se x ho rm ones (fo llicu line). U n d er th e in f lu ence of te s to s te ro n e th e sa liv a ry g lan d of th e fem ales ta k e s on th e ty p e of th e m a le s tru c tu re , w h ile th e sa liv a ry g lan d of th e m ales changes to th e fem a le ty p e u n d e r th e in flu en ce of fo llicu line . T h e re s u lt o b ta in ed by th e e x p e r im e n t m ad e it possib le fo r th e a u th o r to conclude th a t d if fe re n t fa c to rs w h ich d ep en d on th e sex of th e an im al, e x is t in th e sa liva .

T a k i z a w a observed th a t th e re is an in te rd e p e n d e n c e b e tw een th e fu n c tio n of th e sa liv a ry g lands an d th e bony sy s tem ( T a k i z a w a , 1954; G a b e , 1950).

In 1940 F u k u s h i m a fo und co n sid e rab le changes in th e te e th of m ice an d ra ts w ith induced sa liv a defic iency ( O g a t a , 1955). I s h i i (1944) d esc r ib ed th e se changes by m eans of h is to log ica l m e th o d s ( O g a t a , 1955). T he sam e y e a r O g a t a iso la ted a fra c tio n of ac tiv e su b s tan ce fro m th e sa liv a ry g lands of a b u ll, and in je c te d young ra ts w ith th is su b s tan ce . In th is e x p e r im e n t he observed an in c rea se in h a rd tissu es and a red u c tio n in th e lev e l of ca lc ium in th e b lood se ru m . 11 o an d M i z u t a n i ca lled th is su b s tan ce , w h ich w as in th e n a tu re of a p ro te in so lub le in w a te r , p a ro tin (I t o, 1954).

In 1949 11 o o b ta in ed c ry s ta llin e fo rm a tio n s fro m p a ro t in , an d in 1952 to g e th e r w ith O g a t a gave a d e sc rip tio n of th is su b s tan ce , w h ich w as acknow ledged to be specific h o rm one of th e p a ro tid sa liv a ry g lan d ( O g a t a , 1955).

G a b e (1950) ob serv ed a tro p h y of th e sa liv a ry g lands in w h ite r a ts w h e re th e p itu ita ry g land had p rev io u sly been rem o v ed by o p era tio n . B e s i d e s a nd A h a - z a 1 i show ed th a t a f te r excis ion of th e sa liv a ry g lan d s th e n u m b e r of ac idoph ilous cells in th e p itu ita ry g lan d in c reases . T hese cells d is a p p e a r a f te r p a ro tin is ad m in is te red .

R a y n a u d (1954 b) found th a t exc is ion of th e su p ra re n a l g lan d in r a ts causes a tro p h y of th e p a ro tis .

V a l e r i (1954 a, b) gave ra ts te s to s te ro n and th e n e x am in ed its e ffec t on th e sa liv a ry g lands. H e fo und th a t th e c e llu la r n u c le i en la rg ed in th e sec re tin g sec tions. As is w ell kno w n , e n la rg e m e n t of th e c e llu la r n uc le i is b o th a te s t and an in d ex of th e in c rea sed ac tiv ity of th e cell. H e ob serv ed d is tin c tly sm a lle r c e llu la r nu c le i in c a s tra te d an im als co m p ared to th e co n tro l an im als .

P a r h o n e t al. (1956) gave ra b b its , an e x tra c t p re p a re d fro m th e p a ro tis of a bu ll and found th a t th e e x tr a c t w as cap ab le of ra is in g th e su g a r co n ten in th e b lood.

P a r h o n e t al. (1955 a) rem o v ed th e p a ro tid sa liv a ry g lan d s of ra ts , th e n e x a m ined th e e ffec t of th e absence of th ese g lan d s on th e te s tic les . T hey fo u n d th a t u n ila te ra l rem o v a l of th e p a ro tis caused co m p en sa to ry a h y p e r tro p h y of th e re m a in in g p a r t of th e g land , b u t d id n o t cau se any changes in th e tes tic le s . O n th e o th e r hand , a f te r b ila te ra l re m o v a l of th e p a ro tis , a d is tin c t red u c tio n in th e p rocesses of sp e rm a to g en esis w e re observed .

G o d e t (1956) fo und an in te rd e p e n d e n c e in th e m ole b e tw e e n th e d e v e lo p m en t of p a ro tis an d th e se x u a l g lan d s . H e re a c h e d th e conclusion th a t th e h e ig h t of th e

.ep ith e liu m of th e se c re to ry vesicles in th e sa liv a ry g lands d ep en d s to a c e r ta in e x te n t on th e h o rm o n e se c re te d b y th e te s tic les .

In re c e n t y e a rs h is to lo g ica l s tu d ie s h av e been p u b lish ed in w h ich use w as m ade of h is to ch em ica l te ch n iq u e o r ra d io -a c tiv e iso topes.

D e w e y (1958) in v e s tig a te d , by m ean s of h is to ch em ica l m e th o d s, th e p a ro tis of the w h ite r a t a f te r th e p itu ita ry g lan d h ad been rem oved . H e fo u n d changes n o t only

218 I. D z ie rzyk ray -R og a lska

in th e v e s ic u la r cells, w h ich becom e sm alle r, b u t also changes in th e co n ten ts o í g lycogen, RNA , PA S , specific e s te ra se and th e su lp h y d ry l g roups.

O g a t a (1955), T a k i z a w a (1954) fo u n d an in c rea se in th e ep ith e liu m of th e th y ro id vesicles a f te r excis ion of th e sa liv a ry g lands in th e w h ite ra t .

S a w i c k i (1961), u sin g J IS1 on ra ts , observed in c reased accu m u la tio n of iod ine in th e th y ro id g lan d of th e an im a ls as a re s u lt of th e rem o v a l of th e sa liv a ry g lands. T he a u th o r fo u n d less dep o sitio n of J 1S1 in th e th y ro id g lands of an im als w h ich had b een g iv en p a ro tin .

S a s a k i e t al., 1953; D z i e r z y k r a y - R o g a l s k a & G u t s z e (1963) using p h o sp h o ru s P 32, ex am in ed th e bones of w h ite m ice a f te r th e sa liv a ry g lands h ad been rem oved an d p a ro tin ad m in is te red .

No cells w h ich could be re sp o n sib le fo r th e d u c tle ss sec re to ry ac tiv ity have , as y e t, b een found in th e sa liv a ry g lands . G o l i a n i t z k y (1924) considered th a t th e h o rm o n a lly ac tiv e cells a re th e b a sk e t cells. D u rin g th e la s t 30 y ea rs n u m ero u s h is to logical, physio log ica l an d b iochem ica l s tu d ie s of th e sa liv a ry g lan d s h av e been p u b lish ed by th e Ja p a n e se schoo l (I t o, 1954, 1960; R a u c h , 1959; E n g l i s h , 1960; F l e m i n g , 1960; F u j k i e t al., 1960; G o d l o w s k i e t al., 1960; L e v i - M o n - t a l c i n i e t al., 1960; M y a n t , 1960; S c h n e i d e r e t al., 1960; S r e e b n y , 1960; A s a n o , 1961a, b ; B a b a e v a e t al., 1961; I n n e s e t al., 1961; Q u i n t a r e l l i e t al., 1961). A s show n by th e se s tu d ies , th e sa liv a ry g lands m u s t be inc luded in th e d u c tless g lands g roup , b u t th e m a jo r ity of th e a u th o rs do n o t g ive th e ir o p in ion as to th e p lace in th e sa liv a ry g lan d s in w h ich th e h o rm o n e is fo rm ed . A ccord ing to O g a t a , th e sec re tio n of th e sa liv a ry g lands fo rm ed in th e vesicles, flow s th ro u g ii th e in te rc a la r duc ts to th e s t r ia te d d uc ts and is th e re p a r t ly ab so rb ed to g e th e r w ith th e ho rm o n e by th e ep ith e lo id cells lin in g th e lu m en of th e tu b es . N ex t th e sec re tio n reach es , th o u g h th e se cells, th e ly m p h a tic a re a s su rro u n d in g th e s t r ia te d duc t. In th e cells lin in g th e lu m en of th e s tr ia te d d u c ts th e re is a specific sy s tem of m ito ch o n d ria , and cana ls ly ing p a ra lle l to th em , w h ich is in d ica tiv e o f the ab so rp tiv e cap ac ity of th ese cells. T he ab so rp tiv e ac tiv ity of th e cells described ( O g a t a , 1955) w as a lso d e m o n s tra te d by in je c tin g p ro te in so lu tio n in to th e m ain du c ts of th e p a ro tis . A t th e sam e tim e , th a t sam e p ro te in so lu tio n w as in jec ted su b - cu taneously , an d th e ra p id ity of th e a p p ea ran ce o f th e p ro te in ad m in is te red in th e blood w as com pared . I t w as found th a t ab so rp tio n of th e p ro te in in je c te d in to th e ducts , ta k e s p lace s ix tim es m ore ra p id ly in co m p ariso n w ith th e ab so rp tio n of th is so lu tion g iven su b cu tan eo u sly .

K r a c h t (1960), w ho gave th e e x p e rim e n ta l an im a ls p a ro tin , found changes w ith in th e p a ro tid sa liv a ry g lan d s w h ich led to th e conclusion th a t th e v esicu la r cells a re connected w ith d u c tless sec re to ry ac tiv ity .

11 o (1960) in an a ly s in g th e a c tiv ity of d if fe re n t sa liv a ry g lands found th a t d u c t- lees s e c re to ry ac tiv ity is e x h ib ite d only by th e p a ro tid and su b m a x illa ry sa liv a ry g lands . I t seem ed, in th a t a u th o r ’s op in ion , th a t th e su b lin g u a l sa liv a ry g lan d s do n o t p a r t ic ip a te in sec re to ry en d o crin e ac tiv ity . P a ro t in is a h o rm o n e p ro d u ced ch ie fly in th e p a ro tis .

I t is c lea r from th e l i te r a tu re on sa liv a ry g lands re fe rre d to above — th a t these g lands sec re te a h o rm o n a lly ac tiv e su b s tan ce cap ab lae of ca lc ify ing h a rd tissues and of a lte r in g th e leve l of ca lc ium in th e blood se rum .

As w e k n o w th e p a ra th y ro id g lands p lay a d o m in a n t p a r t in -phosphorus-ca lc iu rn reg u la tio n .

I t is assum ed th a t th e p a ra th y ro id g lands belong to th e g ro u p of d u c tless g lands w ith a fu n c tio n m a in ly m etabo lic , th a t is, g lands , th e ac tiv ity of w h ich w ou ld ap p ea r

P a ro t id sa l iv a ry g lands an d p a ra th y ro id g lands 219

to depend on th e lev e l of c e r ta in su b s tan ces in th e body flu id s. (A s tim u lu s to these g lands is th e red u ced leve l of th e su b s tan ces re g u la te d by th em e.g. h y p e rfu n c tio n of th e s u p ra - re n a l co rtex is caused by too low a lev e l of n a tr iu m ).

T he s lig h te s t q u a li ta t iv e or q u a n ti ta t iv e f lu c tu a tio n s of ion ized ca lc ium a re c a p ab le of causing se rio u s d is tu rb an ce s in th e sy s tem th e re fo re , an ex p ress io n of th ese p rocesses shou ld be m orpho log ica l changes ta k in g p lace in th e p a ra th y ro id g lands ( M o r g a n , 1936; F o r s t e r , 1943 a, b , 1946; A b d e r h a l d e n , 1952; H a r v a t , 1954; E g e r , 1954; B a r g m a n n , 1939; E 1 k i n t o n , e t al., 1955; L i c h w i t z e t al., 1955; T a i m a g e e t al., 1955; K r o o k , 1957; W e y m o u t h , 1957; G r a u , 1958; L a n g e , 1961; F u j i i, 1960 a, b; H a r t w i g, 1962).

I t is h o w ev er, g en e ra lly k n o w n th a t e v a lu a tio n of p a ra th y ro id g land fu n c tio n by m orpho log ica l m ethods is v e ry d ifficu lt.

T he ch ie f c lassif ica tio n of th e cells of th e p a ra th y ro id g lands is b ased on th e s tu d y m ade by S a n d s t r o m (1880), w ho w h ile d esc rib in g th e p a ra th y ro id g lan d s w as th e f i r s t to , observe tw o types of cells — cells r ich in cy top lasm an d cells w ith a sm a ll am o u n t of cy top lasm . T he n e x t w as W e l s h (1889) w ho in d esc rib in g th e cells r ic h in cy top lasm te rm e d th em m a in cells, and th e second k in d , poor in cy to p lasm , o x y p h il cells. G e t z o w a (1907), d iv ides th e ch ief cells of th e p a ra th y ro id g lands in to lig h t (W asserhelle Z ellen) and d a rk (R osaro te Z ellen) acco rd ing to th e a ff in ity of th e cy to p lasm to dyes. In ad d itio n she d is tin g u ish es ac id iph ilous cells and sy n c y tia l cells.

B o b e a u (1914) fo u n d in th e h o rse a co n sid e rab le v a rie ty of cell types, such as basic cells, n o rm a l cells, lig h t and d a rk cells, la rg e g ra n u la r o r co llo idal cells, sy n cy tia l bodies, p ro to p la sm a tic an d spong iocy tes.

In 1935, C a s t l e m a n a nd M a l l o r y d is tin g u ish ed b e tw een p a ra th y ro id cells, d iv id in g th em in to d a rk and lig h t cells, w h ich u n ite in to a com m on g ro u p of chief cells (no rm al). T hey also d is tin g u ish o x y p h il cells, d iv id in g th e m in to lig h t and d a rk cells. A cco rd ing to th ese au th o rs th e p a re n c h y m a o f th e g lan d consis t m a in ly of d a rk ch ief cells. T he lig h t ch ie f cells fo u n d in th e p e rip h e ry of th e g lan d occur in v e ry sm a ll am oun ts .

C o r r e i a M a d e i r a (1942) took th e c o m p a ra tiv e h is to logy of m a n and m am m als as a b asis fo r c lassif ica tion of th e cells. H is c lassif ica tio n is s im ila r to th a t of C a s t l e m a n a nd M a l l o r y ( C o r r e i a M a d e i r a , 1942— 1946).

W hile th e re is s t i l l c e r ta in ag re e m e n t in th e v iew s of au th o rs as to th e m o rp h o log ical c la ss if ica tio n of th e g la n d u la r cells of th e p a ra th y ro id g lands , th e q u es tio n of th e o rig in an d fu n c tio n of each ce llu la r ty p e s t il l re m a in s u n ex p la in ed .

S ince th e ap p e a ra n c e of th e f i r s t d e sc rip tio n of th e p a ra th y ro id g lands d iscussion h as co n tin u ed on th e p ro b lem of w h ich of th e cells, ch ie f o r oxyph il, a re th e sec re tin g e lem en ts .

P e p e r e (1906) desc rib ed th e cycle of tra n s fo rm a tio n of th e p a ra th y ro id cells, w h ich lead s fro m th e ch ie f cell to th e o x y p h il se c re to ry ce ll a n d back . H e considered th e o x y p h il cell th e m ost ac tive , an d h e ld th a t it is a changed ch ie f cell ch a ra c te rise d by h igh se c re to ry ac tiv ity (R u c a r t, 1949).

In 1919 B e r g s t r a n d , and la te r H o f f h e i n z (1925) cam e to opposite con clusions: th e y co nsidered th e o x y p h il cell to b e d e g e n e ra te cells d ep riv ed of a se c re to ry fu n c tio n . T h is co ncep t w as to f in d an in c rea s in g ly la rg e n u m b e r of a d h e re n ts am ong h is to log ists , an d in p a r t ic u la r am ong h is to p a th o lo g is ts (R u c a r t, 1949).

B e r g m a n n (1939) considers th a t th e lig h t ch ie f cells and d a rk ch iefs cells a re sec re to ry e lem en ts , w h ile th e o x y p h il cells fo rm ed from th em a re d eg en e ra tin g

220 I. D zie rżykray-R ogalska

in ac tiv e fo rm s, w h ich in th e con tinued process of d eg en era tio n m ay u ndergo fa t ty d eg en era tio n .

A la rg e n u m b e r of a u th o rs (R o s o f, 1934; d e R o b e r t i s , 1940—41; I g n a t i e v a , 1948; R u e a r t , 1949; H a n s l e r , 1953; C z e r s k i , 1952; D z i e r ż y k r a y - R o g a l s k a , 1954; 1958; W i ś n i e w s k i , 1960; H a r a e t al., 1957; 1959; 1962), consider th a t a ll th e cells occu rring in th e p a ra th y ro id g lands a re th e sam e ty p e of cell a t d if fe re n t fu n c tio n a l stages.

R o s o f (1934) in h is inv es tig a tio n s of th e s tru c tu re of th e p a ra th y ro id g lands in r a t s d ep en d in g on age, p reg n an cy and d ie t, s ta ted th a t h is to log ica l changes in th e g lands of d if fe re n t g roups of an im als a re sligh t. T he d iffe rences consist r a th e r in th e a r ra n g e m e n t of th e cells th a n in th e i r k ind . H e d is tin g u ish es in th e ra t , ch ief cells, l ig h t cells and ch rom oph ilous cells, an d considers th a t th e ch ief cells a re fo rm ed fro m lig h t cells. T he la t te r are , he considers, in ac tiv e cells. In th e co n tro l an im a ls th e a u th o r fo u n d th a t only 5°/o — 25°/o of th e cells w e re in th e sec re to ry stage. H e is of th e op in ion th a t th e ce llu la r o rgane lle such as th e G olgi a p p a ra tu s an d chond riom , w h ich a re su b je c t to changes in s tru c tu re d u rin g th e sec re to ry cycle, p lay an ac tive p a r t in c e llu la r secre tion .

C o r r e i a M a d e i r a (1942— 1946) has m ade a la rg e n u m b er of d e ta iled cy to - log ica l s tu d ies b ased on in v es tig a tio n of th e G olgi a p p a ra tu s and d is tr ib u tio n of th e ch ond riom , d iscussing th e e ffec t of th e fix in g flu id used on th e p ic tu re ob ta ined . H e considers th a t only th e d a rk ch ief cells (w hich he te rm s ch ief cells) and th e lig h t ch ie f cells a re of fu n c tio n a l significance. T he n u m b er of lig h t cells en co u n te red in th e g lan d se rv ed h im as a c rite r io n of th e fu n c tio n a l s ta te of th e g land . H e gives an d e ta iled d e sc rip tio n of th e G olgi a p p a ra tu s and chondriom , b u t does no t g ive h is op in ion on th e p a rtic ip a tio n of these o rg an e lle in c e llu la r secre tion . T he au th o r did n o t find a co n s ta n t p o la r position of th e Golgi ap p a ra tu s , e ith e r in re la tio n to connec tive tissu e o r in re la tio n to th e blood vessels. T he v a ria b le s tru c tu re of th e G olgi a p p a ra tu s in th e se c re to ry cycle of th e cells of th e p a ra th y ro id g lands has, also b een d e a lt w ith by a la rg e n u m b er of re sea rch w o rk e rs ( C o u r v i e r e t al., 1922; D i a s - A m a d o e t ai., 1944; de R o b e r t i s e t al., 1940—1941; R u c a r t, 1949).

B e n s 1 y (1947) in v e s tig a te d th e p a ra th y ro id g lands of dogs, from w hich she rem o v ed p a r t of th e g lands, causing h y p e rtro p h y of th e rem a in in g p a rt . T he a u th o r en d eav o u red to fin d p re -se c re to ry g ra n u la r m a tte r w ith in th e cells. In h e r opinion th e y occur in th re e fo rm s: a) vacuoles s ta in in g v e ry fa in tly , b) g ran u les s ta in ed in ten s iv e ly and c) co llo idal globules. She d id not, how ever, find any connection b e tw een se c re to ry fu n c tio n an d th e cy top lasm atic fo rm a tio n s exam ined .

I g n a t i e v a (1948) w ho in v es tig a ted th e p a ra th y ro id g lands of m am m als (dogs, ca ts , ra b b its , g u in ea pigs and w h ite ra ts ) found th a t hom ogeneous cells occur. She is of th e op in ion th a t th e process of th e en d o ce llu la r p ro d u c tio n of th e sec re tio n is m an ife s ted m o rp h o lo g ica lly by th e p resence , in th e cy top lasm , of sm all vacuoles, w h ich g ra d u a lly fu se and fo rm ligh t fields. A fte r com plete d ilu tio n of th e cy top lasm , th e secre tio n flow s in to th e blood vessels. T he cell, w h ich is in th e f in a l s tag e of a ccu m u la tin g th e secre tio n , appears as a com plete ly lig h t fo rm a tio n , g re a tly en la rg ed . I g n a t i e v a considers th a t th e w hole cy top lasm is re sponsib le fo r th e p ro d u c tio n of th e sec re tio n , and no t d if fe re n t o rgano id s such as, fo r exam p le , ch ond riom o r th e G olgi ap p a ra tu s .

F o r t i n a (1948) in d u ced rick e ts in ra ts and found th a t o x yph il ceils ap p ea red , w h ich a re k n o w n to occur ra re ly in ra ts . T he a p p ea ran ce of o x y p h il cells, w as in th e a u th o r ’s opinion, ev idence of increased ac tiv ity of th e g land,

P aro t id sa l iv a ry g lands and p a ra th y ro id glands 221

R u c a r t (1949), tak in g co m p ara tiv e h is to logy as h is basis, in tro d u ced h is ow n c lassif ica tio n of types of cells in th e p a ra th y ro id g lands. F ro m th e em bryon ic cell, fro m w h ich th e basic ce ll is fo rm ed , th e au th o r d is tin g u ish ed tw o series of cells d iffe rin g fro m each o th e r m orpho log ica lly and fu n c tio n a lly :

A) L ig h t se r ie s : a) basic cell, b) n o n -s ta in in g cell w ith a sm all n u m b e r of vacuoles,c) n o n -s ta in in g cell w ith a la rg e n u m b e r of vacuoles, d) lig h t cell.

B) D a rk se ries: a) basic cell, b) ch rom oph ilous cell, c) ox y p h il cell, fro m w hich th e g ra n u la r ox y p h il cell or th e o x yph il cell w ith vacuo les m ay be fo rm ed .

R u c a r t on th e basis of h is ow n re se a rc h reach ed th e conclusion th a t th e p a ra th y ro id g lands sec re te a t le a s t tw o horm ones. O ne of th em w ould be fo rm ed in th e vacuoles (ligh t series), and th e p re c u rso r of th e second w ould be fu ch s in o p h il g ran u le s (d ark series). A ccord ing to th is au th o r b o th se rie s of cells occur in the n o rm ally fu n c tio n in g g lands of h ig h e r m am m als. In th e low er m am m als the p a ra th y ro id g lands a re fo rm ed ch iefly of basic cells, and it is only u n d e r specia l cond itions (p regnancy , ex p e rim en t) th a t cells a p p ea r w h ich can be a llo ca ted to th e lig h t or d a rk series. In ad d itio n , he fo und th a t th e o x y p h il cells can possess sec re to ry ac tiv ity .

H a n s l e r (1953) using ca ry o m etr ic m ethods, in v es tig a ted th e p a ra th y ro id g lands of w h ite r a ts su b jec ted to d if fe re n t ex p e rim en ts , consis ting in th e s tim u la tio n and in h ib itio n of th e g lan d fun c tio n . T he a u th o r no tices th a t in h ib itio n of th e g lan d fu n c tio n w as m an ife s ted by a red u c tio n in th e size of th e nuclei. T he nucle i becam e en la rg ed in th e s tim u la te d g land . S im ila r m ethods of re se a rc h w ere u sed by o th e r au th o rs , w ho a re of th e op in ion th a t c a ry o m etric m ethods giVe m ore re lia b le re su lts in in v es tig a tio n s on g land fu n c tio n th a n m orpho log ica l m ethods (E d e r e t al., 1955); H a d 1 e r e t al., 1962).

E d e r & H a r t l (1955) g ive an ex ac t c lassif ica tio n of p a ra th y ro id cells and th e ir ev o lu tio n a ry capab ilitie s . By s tim u la tin g th e p a ra th y ro id g lands th ey in c reased th e size of th e nuclei, a f te r w h ich th ey d e fined th e n u c lea r-cy to p la sm a tic ra tio . On th e basis of th e ir ex p e rim e n ts th ey en d eav o u red to d e te rm in e th e connection be tw een th e ty p e of cell and its fu n c tio n , using m orpho log ica l and s ta tis tic a l m ethods. T he m orpho log ica l o b se rv a tio n s of th e a u th o rs W ere concerned w ith th e a p p ea ran ce of th e nuclei. On th e basis of s tu d ie s m ade by A l t m a n (1955) w ho fo und th a t p ro te in (g lobulin) m etabo lism in th e cell is connected w ith th e changes tak in g p lace in th e n uc le i, th e au th o rs d iv id ed th e nucle i o ccu rring in th e cells of th e p a ra th y ro id g lan d s in to fo u r types:

1) nucle i, w h ich co n ta in nucleo li s itu a te d c e n tra lly (resting nuclei),2) nucle i, w h ich co n ta in nucleo li s i tu a te d n e x t to th e n u c lea r m em b ran e (usually

th e se a re la rg e r n uc le i p re p a r in g to sec re te n u c leo la r su b s tan ce to th e cy top lasm ),3) n u c le i v isu a lly a lm o st em pty , w ith o u t d is tin c t ch ro m a tin e su b stan ce ,4) n uc le i in w h ich nucleo li an d ch ro m o cen tres can be seen s itu a te d u n d e r th e

n u c le a r m e m b ra n e (type 3 and 4, are nucle i e x h au s ted by in ten s iv e se c re to ry ac tiv ity ).

A t th e sam e tim e , th ey m ad e a s ta tis tic a l ana ly s is of th e 4 types of c e llu la r nucle i described above, using th e C halk ley m ethod , co u n tin g ab o u t 1000 c e llu la r nucle i in one g roup . O n th e b as is of s ta tis tic a l analy sis th ey reach ed the conclusion th a t e n la rg e m e n t of th e n uc leus is a s ig n of its ac tiv ity , w h ile its red u c tio n in size ind ica te s a d ecrease in c e llu la r fu n c tio n . T hese au th o rs , ta k in g v a ria tio n s in th e size of th e nucle i as a basis, consider o x y p h il cells to be in ac tiv e cells, d iv id in g th em in to tw o g ro u p s; d a rk cells and lig h t cells. T hey a re of th e opin ion th a t th e lig h t oxyph il cell is fo rm ed from th e d a rk o x yph il cell.

T h I. D z ie rzykray -R oga lska

In re c e n t y ears m an y stud ies have ap p ea red concern ing th e h is tochem icalin v es tig a tio n of th e p a ra th y ro id g lands (P e a r s e e t al., 1958; S a m n a z z a r i e tal., 1959; W a l t h a r d , 1960; F u j i , 1960; Y a n g , 1960; Z a w i s t o w s k i , 1962).

T r e m b l a y e t al., show ed in 1959, using th e h is tochem ical tech n iq u e , th a to x yph il cells in th e p a ra th y ro id g lands of hum ans and m onkeys a re d is tigu ished byin ten s iv e ac tiv ity of o xygena ting enzym es (DPND, TPN D , succinic acid d eh y d ro genase). T he re su lts ob ta in ed suggest th a t th e o x y p h il cells a re in ten siv e ly fu n c tio n ing cells. T his does n o t m ean th a t th e ir sec re to ry ac tiv ity is g re a te r , i t is possib le th a t th e oxyph il cells h av e sim ply accum ula ted eno rm ous energy . T h e ir fun c tio n and sign ificance , how ever, h av e no t as y e t been th o ro u g h ly in v es tig a ted .

L ite ra tu re on th is p rob lem show s th a t th e re a re m an y e x p e rim e n ta l stu d ies on th e su b m a n d ib u la r sa liv a ry g lands and th e ir connection w ith o th e r en docrine g lands. As y e t, how ever, l i t t le a tten tio n has been p a id to the p a ro tid sa livary g lands and th e changes ta k in g p lace in them u n d e r the in flu en ce of th e fu n c tio n in g in o th e r d uctless g lands. N ot m an y au th o rs have d ea lt w ith th e changes in th e endocrine g lands caused by e x p e rim e n ta l hypo func tion or h y p e rfu n c tio n of th e p a ro tid sa liv a ry g lands (H ypofunction of th e sa liv a ry g lands — asia lad en ism , is caused by th e ir p a r t ia l or to ta l rem o v al, h y p e rfu n c tio n — h y p eras ia lad en ism , by th e tr a n sp la n ta tio n of th e g lands or by in jec tin g paro tin ).

A lth o u g h th e re is an enorm ous am o u n t of l i te r a tu re on th e p a ra th y ro id g lands, and th e m orpho log ica l and h is tochem ical changes w ith in th ese g lands u n d e r th e in fluence of d if fe re n t fa c to rs hav e been th o ro u g h ly in v es tig a ted , y e t I h av e not e n co u n te red s tu d ie s dealing w ith th e changes ta k in g p lace in th em u n d e r th e In fluence of h y p e rfu n c tio n or hypo function of th e sa liv a ry g lands, a p a r t from the sh o r t re fe re n c e m ade to them in th e w ork by O g a t a (1955).

III . M A TER IA L, M ETHODS AND TEC H N IQ U E

T he m a te r ia l used in th e p re sen t study consisted of 185 young w h ite ra ts (sexually m a tu re m ales) w eigh ing from 130— 160 g., w hich w ere k e p t on a s ta n d a rd g ra n u la te d d ie t.1)

T he an im als w ere d iv ided in to IV groups. E ach g ro u p w as su b jec ted to d if fe re n t ex p e rim en ts :

In g roup I, an inves tig a tio n w as m ade in to th e e ffec t of rem oval of th e p a ra th y ro id g lands on th e m orpho log ica l p ic tu re of th e sa liv a ry g lands of th e w h ite ra t.

In g ro u p II, an in v es tig a tio n w as m ade in to th e e ffec t of th e rem o v a l of th e p a ro tid s a liv a ry g lands on th e m orpho log ical p ic tu re of th e p a ra th y ro id g lands.

In th e an im als in g roup II I th e m orpholog ical p ic tu re of th e p a ra th y ro id g lands and p a ro tid sa liv a ry g lands w as in v es tig a ted a f te r th e a d m in is tra tio n of paro tin .-)

In g ro u p IV, P 32 w as used to inves tig a te th e e ffec t of rem o v a l of th e sa liv a ry g lands and doses of p a ro tin on th e bones of th e ra ts .

In each of th e above groups som e of th e an im als w ere used as con tro ls.T he an im als w ere anaesth e tized w ith e th e r and th e n d issec ted , th e p a ro tid

sa liv a ry g lands and p a ra th y ro id g lands being ta k e n fo r ex am in a tio n . O ne sa liv a ry g land and one p a ra th y ro id g land w ere alw ays fixed in L iso n -V o k aer flu id , th e o th er,

J) G ra n u la te d fo d d er fo r ra ts (In s titu te of M edicine, W arsaw ): W heat m ash 19°/o, b a rley m ash 19°/o, oa t m ash 19°/o, w h ea t b ra n 20°/o, fish m ea l 8%, sk im m ed pow dered m ilk 8°/o, fo d d e r y eas t 5%, casein l°/o, v itam in m ix tu re l°/o.

2) T he a u th o r is ex trem e ly g ra te fu l to th e f irm of T eikoku Mfg. Co, Tokio, fo r su p p ly in g th e p a ro tin used in th e p re sen t s tu d y , free of charge.

P a ro t id sa l iv a ry g lands and p a ra th y ro id g lands 223

d ep en d in g on th e la te r p rocedu re , w as fix ed in one of th e fo llow ing flu id s: B ouin, H elly , A oyam o, su b lim a te , C arnoy n e u tra l fo rm ol. T he p re p a re d g lands w ere soaked in p a ra ff in , and sec tions 5 m icrons th ick w ere n e x t s ta in e d by th e fo llow ing m ethods:

a) h a e m a to x y lin -eo s in e — to o b ta in n o rm a l specim ens fo r ex am in a tio n ,b) th e A zan and M allory m ethod ,c) B est’s m ethod — to rev ea l th e p resen ce of glycogen,d) PA S m ethod — to d iscover th e p resen ce of m ucopo ly saccharides (Me M a n u s ,

1948),e) M u lle r’s m ethod , as m od ified by G r a u m a n n an d C 1 a u s s — using colloidal

iro n — to d iscover th e p resence of acid m ucopo ly saccharides (P e a r s e, 1957; G r a u m a n n e t al., 1958),.

f) F eu lg en ’s m eth o d using S ch iff’s re a g e n t — to e s tim a te th e d eg ree of p o ly m erisa tio n of deso x y ry b o n u c le in acid (DNA) — ( C a s p e r s s o n , 1950; K r y g i e r, 1940),

g) B ra c h e t’s m ethod (m ix tu re of p y ro n in B and m e th y l g reen) as m odified by G o d l e w s k i & V o r b r o d t (1954), u sing perch lo ric acid to d iscover th e p resence of R N A (B r a c h e t, 1957),

h) L ag u ess’s m ethod — to d iscover th e p resence of a rg en to p h ilo u s fib res (R o- m e i s, 1953),

i) R eg au d ’s m ethod (ferrous haem .) — to d iscover th e p resence of ce llu la r chond riom ,

j) sections o b ta in ed a f te r fix ing m a te r ia l in A oyam o flu id w ere used to d iscover th e p resence of th e G olgi ap p a ra tu s .

IV. E F FE C T OF TH E R EM OV AL OF T H E PA R A T H Y R O ID G LA N D S ON TH E M O R PH O L O G IC A L P IC T U R E OF TH E SA LIV A R Y G LA N D S OF T H E W H IT E RA T

F ifty sexually mature male w hite rats were used for the experim ent, in forty of which the parathyroid glands were rem oved by operation. The remaining 10 rats were used for control purposes, control group A consisting of 5 rats left intact, and group B, 5 rats on which a blank operation was performed. The rats deprived of their parathyroid glands w ere killed at different intervals after-the operation.

1. Operation technique

T he ra ts w ere an aes th e tiz ed w ith e th e r and placed on th e ir backs on a spec ia l o p e ra tin g ta b le (F a r i s e t al., 1949). A n incision w as m ad e in to th e sk in along th e os h yo id eu m to th e m a n u b r iu m s tern i. T he sa liv a ry g lands w ere p re p a re d u p w ard s, m u sc u li s te rn o -c le id o -m a sto id e i p re p a re d sidew ays and th e sh o r t m uscles of the neck w ere cu t in f ro n t of th e tra c h e a . A fte r th e cu t m uscles h ad been d ra w n a p a r t by hooks th e tra c h e a and th y ro id g lan d cou ld be seen. T he p a ra th y ro id g lands could be seen in th e u p p e r poles of th e th y ro id , m o re to th e back , in th e p lace w h e re th e u p p e r th y ro id a r te ry p e n e tra te s in to th e p a ren ch y m a of the g land . D u rin g th e f irs t p eriod of th e ex p e rim e n ts th e p a ra th y ro id g lands w ere rem o v ed from th e an im als to g e th e r w ith th e su rro u n d in g tissu e of th e g land , w hich caused fre q u e n t h a e m o rrh ag es from th e u p p e r th y ro id a r te ry (G o u d a 1, 1955; S n e l l , 1956). On th is accoun t in la te r o p e ra tio n s, a f te r th e p a ra th y ro id g lan d s had been rev ea led , they w ere

224 1. D zierzykray -R ogalska

g ra sp ed w ith sm a ll fo rceps and th e pedunc le of th e g land liga ted , n ex t ex c is in g th e p a re n c h y m a of th e g land , as fa r as possib le in its en tire ty . C lin ical sy m p to m s of in su ffic ien cy of th e p a ra th y ro id g lands occured w ith u n ifo rm in te n s ity a f te r th e o p e ra tio n h a d been perfo rm ed in th e above w ay. A fte r sp r in k lin g p en ic illin in to th e w o und , th e sk in w as sew n up w ith con tinuous c a tg u t su tu re .

B efo re th e an im a ls w ere k illed th e ir blood sam p les w ere ta k e n by card iac p u n c tu re in o rd e r to define th e calcium c o n ten t in th e se ru m . T he ca lc ium level w as ca lc u la ted by m ean s of th e flam e p h o to m e te r (M odel I I I m ade by th e f irm cf C arl-Z e iss) , p rev io u sly d raw ing th e cu rv e of ca lib ra tio n fo r calcium .

D u rin g d issec tion cu ttin g s w ere ta k e n from th e p a ro tid sa liv a ry g lands in five p e rm a n e n t p laces. A t th e sam e tim e th e th y ro id g land w as rem oved , a f te r w h ich i t w as cu t in series in o rd e r to check th a t th e p a ra th y ro id g lands had been c o m p le te ly excised .

C o n t r o l a n i m a l s — (Controls A and B) calcium level in the blood serum was on an average 10 mg%.

The parotid salivary gland of control anim als is a vesicular-serous gland.

The histochem ical tests made reveal the presence of a fairly considerable amount of RNA in the basic parts of the vesicular cells (Fig. 1, P late X). No glycogen w as discovered. Staining w ith colloidal iron revealed the presence w ith in the vesicular cells of parotis of a large amount of acid m ucopolysaccharides. W ithin the cells of the intercalar ducts slight PAS-I- granular m atter was observed. The striated ducts formed of cylindrical cells arranged very regularly side by side, did not give any of histochem ical reactions applied. When staining by the Regaud m ethod was used, parallel lines of striation became visible w ith in the striated saliva ducts in the basic part of the cells, which according to certain authors is cellu lar chondriom (Fig. 2, Plate X).

G r o u p I — animals killed during the first w eek after operation. Calcium level in the blood serum was on an average 7.1 mg%.

The structure of the secretory sections of the gland is similar to that of the glands of the control animals. W ithin the interlobular ducts an apparently double-layered epithelium, the internal layer of which flakes off into the lum en of the duct in the form of a ribbon is observed (Fig. 3, Plate X). D istinct differences in comparison with the control occur after applying histochem ical tests. A marked decrease is observed in the RNA

2. Description of material

The anim als in this group were divided as follows: C o n tro l A — in ta c t an im als C o n tro l B — an im als a f te r b lan k opera tionG ro u p I — an im a ls k illed du ring th e f i r s t w eek a f te r op era tio nG ro u p I I — an im als k illed from 2—4 w eeks a f te r o p e ra tio nG ro u p I I I — an im a ls k illed in th e 6th or 7th w eek a f te r ap e ra tio n

5 an im als 5 an im als

15 an im als 15 an im a ls 10 an im als 50 an im alsT o ta l

P aro t id sa l iv a ry g lands and p a ra th y ro id g lands 225

contents at the base of the glandular cells. W hen colloidal iron is used the vesicles of the salivary gland also stain, but very delicately, scarcely perceptibly.

The anim als in this group ceased eating, became restless and exhibited excessive excitability as early as the day follow ing the operation. In the follow ing days the occurrence of fibrotic tw itches of the m uscles lasting for several m inutes was observed. Attacks of convulsions recurred in the operated anim als w ith d ifferent degrees of intensity.

G r o u p II — animals killed in the second, third or fourth w eek after operation. The average figure for the calcium level in the serum was 7.5 mg%.

The changes are the most distinct in this group of animals. In anim als w hich w ere killed during one of the recurring attacks of convulsions, in addition to the norm ally formed lobes of the parotid glands there are lobes of very greatly altered structure (Fig. 4, P late X). The change in structure consists in the glandular cells being greatly reduced. In this w ay vesicles w ith a fairly large lum en are formed, which makes them som ew hat sim ilar to the vesicles of the thyroid gland (Fig. 5, P late XI). In addition the cytoplasm of the cells is very strongly vacuolised, and their nuclei becom e pycnotic and are thrust towards the base (Fig. 6). The lum en of such a new-form ed vesicle is filled w ith the granular secretion. The striated ducts have a sim ilar appearance (on account of the large lum en and flattened epithelium ), and can be recognised by m eans of the irregular shapes and greater amount of connective tissue w hich surrounds them.

In the later w eeks after the operation (3rd and 4th) connective tissue w ith a large amount of fatty cells can be observed to penetrate betw een the changed v isicles (Fig. 7, 8, P late XI). Vacuolisation of the vesicular cells disappears during this period, the cells becom e flatter and their nuclei pycnotic. A sim ilar process takes place in the striated ducts. The secretion observed in the lum en of the vesicles also slow ly disappears. In the gland changed in the above w ay all the histochem ical tests gave negative results (RNA, PAS, M uller’s m ethod, B est’s method).

G r o u p III — anim als killed in the 6th or 7th w eek after the operation. The calcium level in the serum was on an average 8.1 mg%.

The parotis in the anim als of this group is built norm ally of lobes, differing in that the vesicular cells and their nuclei are sm aller and that num erous m itoses, which are not observed in the other groups of anim als, occur w ith in the striated ducts. In addition to the norm ally built lobes of the salivary gland, aglom eration of fatty tissue is observed to have (Fig. 9, P late XI), formed in place of the glandular lobes which have disappeared, the striated ducts how ever remaining.

226 I. D zie rzykray -R oga lska

3. Results

1. The parotid salivary glands of rats after excision of the parathyroid gland exhibit distinct morphological and histochemical changes.

2. As early as the first week after the operation flaking of the epithelial ribbons is observed into the lum en of the interlobular ducts. The amount of RNA and acid mucopolysaccharides within the glandular ceils decreases.

3. From the second to the fourth week after the operation great changes are observed w ithin certain lobes. The glandular cells are reduced, and their protoplasm undergoes intense vacuolisation. The lum en of the vesicles increases, making them similar to the vesicles of the thyroid gland. The nuclei of these vesicular cells become pycnotic. Secretion appears in the lum en of the vesicles and striated ducts and disappears in the fourth week.

4. In the sixth and seventh week the glandular lobes are built of vesicles, the cells and nuclei of which appear to be smaller. The parotis of anim als from this period give very faint histochem ical reactions (RNA and colloidal iron). Numerous mitoses appear within the epithelium of the striated ducts. Lobes of fatty tissue, w ith remaining striated ducts running through them, are encountered in addition to the glandular lobes.

5. The results of the investigations made justify the statem ent that after removal of the parathyroid glands, morphological changes occur in the parotid salivary glands of the rat indicating the disappearance of glandular tissue in favour of fatty tissue.

V. E FFE C T OF TH E REM OVAL OF TH E PA R O TID SA LIV A RY G LA ND SON TH E M O R PH O LO G IC A L P IC T U R E OF TH E PA R A T H Y R O ID G LA ND S

Seventy sexually mature male w hite rats were used for the experim ent, of which the parotid salivary glands w ere removed in 60 animals, and 10 anim als w ere used as controls. The animals deprived of the parotid salivary glands were killed at d ifferent periods of tim e after the operation, and the parathyroid glands were then taken, fixed and stained according to the methods already discribed.

1. Operation technique

T he ra ts w ere an aesth e tized and o p era ted on in a position s im ila r to th a t desc rib ed in th e case of th e rem oval of th e p a ra th y ro id g lands. A len g th w ise in c ision m ad e in to th e neck revea led th e sa liv a ry g lands. P re p a ra tio n of th e p a ro tid s a liv a ry g lands p re se n te d g re a t tech n ica l d ifficu lties, since in th e r a t th ey are s itu a te d in close connection w ith th e deep vessels of th e neck, w h ich can easily be dam ag ed d u rin g th e opera tion . In add ition th e ir recesses reach th e angles of th e


m an d ib u le and p a rtly su rro u n d th e e x te rn a l e a r ducts . T he L o e v e n t . h a l g lands, w h ich h ad to be le f t, in ta c t, w e re o ften closely connec ted w ith th e sa liv a ry g lands ( W a l k e r , 1958; M i n a m i e t al., 1959; D z i e r z y k r a y - R o g a l s k a e t al., 1960; L e e s o n , 1960). R em oval of th e p a ro tid sa liv a ry g lands w as th e re fo re begun by se p a ra tin g th e blood vessels of th e neck , th e n th e L o e v e n t h a l g lan d w as re leased , th e recesses of th e p a ro tis se p a ra te d and th e p a ro tis rem oved in one block. A fte r rem o v a l of th e sa liv a ry g lan d s th e sm all b leed in g vessels w e re lig a ted , th e w ound sp r in k led w ith p en ic illin , an d th e sk in f irm ly sew n up w ith con tinuous ca tg u t su tu re .


The animals in this group w ere divided as follow s:

C on tro l A — an im als le f t in ta c t 5 an im alsC on tro l B — an im als on w hich a b la n k o p e ra tio n w as p e rfo rm ed 5 an im alsG roup I — an im als k illed d u rin g th e f i r s t w eek a f te r th e o p e ra tio n 15 an im alsG roup II — an im als k illed in th e second w eek a f te r th e o p e ra tio n 15 an im alsG roup I I I — an im als k illed in th e th ire d w eek a f te r th e o p e ra tio n 15 an im alsG ro u p IV — an im als k illed in th e 4 th or 5 th w eek a f te r th e o p e ra tio n 15 an im als

T o ta l 70 an im alsC o n t r o l s A and B.The parathyroid glands of rats are built of tw o kinds of cells: a) dark

cells, b) light cells, (a third kind of cells — the oxyphil cells — appear, as already stated only under special conditions).

The dark cels [corresponding to the dark cells of R o s o f (1934) and B e n s 1 e y (1947), chief cells of R u c a r t (1949) and the second type of cells given by C z e r s k i (1952)]. These cells are most num erously represented and it is they w hich form the parathyroid gland. Their size varies from 3— 10 microns. The lim its of these cells are not distinct and their shape m ay vary from m ultilateral to pyram idal and irregular. The cellular cytoplasm is filled w ith sm all densely arranged granular matter. The nuclei of the cells exh ib it considerabe polym orphism. They can be variform nuclei, spherical or even elongated, and large. These nuclei are surrounded by a distinct nuclear membrane, and certain of them stain hyperchrom atically (Fig. 10, P late XII).

The Golgi apparatus in these cells is fairly abundant, most often lying on the apical pole of the cell (we take that part of the cell supported by blood vessels (to be the base).

In addition to the dark cells described, there are light cells in each gland forming the peripheral part of the parathyroid gland. The light cells are larger than the dark, their lim its are more clearly v isib le and the cellular nuclei, usually large, have a distinct chromatin stroma and tautly stretched thin nuclear membrane. The cytoplasm of these cells is filled w ith small less densely distributed granular m atter and vacuoles of d ifferent size, which together give the im pression of light cytoplasm . It

228 I. D zie rzykray-R ogalska

m ust be em phasised that this picture depends to a great degree on the fixing m edium used. The cytoplasm of cells fixed in Lison-Vokaer fluid retains the sm all, loosely scattered, granular matter and as a result is slightly darker. A fter fixing in Bouin fluid the granular matter referred to disappears com pletely and the cells then give the impression of light, em pty, and vacuolised formations. These cells are often arranged in clumps. The histochem ical methods used did not permit of discovering significant d ifferences betw een the two types of cells.

The pictures of the parathyroid glands of control groups A and B do not d iffer from each other.

G r o u p I — parathyroid glands of rats killed during the week fo llow ing rem oval of the parotid salivary glands.

No perceptible morphological changes are found in the parathyroid glands of this group of animals. Only the presence of a dual kind of nuclei in the dark cells is distinct, particularly after using the Feulgen reaction to DNA. Som e of them are larger, sometimes vesicular w ith delicately scattered chromatin, others smaller, irregular in shape and more hyper- chromatic.

G r o u p II — parathyroid glands of rats killed tw o weeks after removal of the parotid salivary glands.

The parathyroid glands w ere still built of two kinds of cells: dark and light cells. As in the case of the glands in the control animals, the dark cells lie ch iefly in the central parts of the gland but occupy a far sm aller part of it. On the other hand the periphery of the gland, formed of light cells, undergoes considerable growth.

The cells (light) situated peripherally are very often arranged in clumps or pseudo-vesicles w ith a very small lumen in the middle (Fig. 11, Plate XII). The cytoplasm of these cells is lighter in comparison with analogical cells observed in the control glands. The chromatin stroma is clearly visible in the m ajority of the irregularly oval-shaped nuclei.

A characteristic feature is the occurrrence in the cells of the parathyroid glands of this group of animals of both mitoses (Fig. 12, P late XII) and amitoses (Fig. 16, P late XIII). The phenomenon is almost totally unencountered in the control animals. In addition, both in the central parts and in the peripheral glands, ’’restlessness” of the nucleus is observed, m any of the nuclei possessing a thickened, and som etim es folded nuclear membrane.

A frequent phenom enon here is also the occurrence of large nucleoli situated in the im m ediate vicinity of the nuclear membrane, which gives the impression of the nucleolar material being secreted into the cytoplasm.

The above pictures are repeated in all the glands of this group of animals.

P a ro t id sa l iva ry g lands and p a ra th y ro id g lands 229

The Brachet method made it possible to discover the scanty RNA granular matter scattered over the whole cytoplasm. This granular m atter is often grouped in the apical part of the cell, above the nucleus, in the cells situated peripherally, and in addition the presence of PAS positive granules was detected in these cells (Fig. 13, P late XII).

The Golgi apparatus in the cells is situated in the apical part (the basic part of the cells is supported by the connective tissue or blood vessels) and is composed of thick trabeculae (Fig. 14, P late XIII).

G r o u p III — parathyroid glands of rats killed in the third week after removal of the parotid salivary glands.

The parathyroid glands of this group of anim als are very sim ilar in appearance to those described in group II. It would, however, seem that more cells w ith P A S + granular matter are encountered in the periphery. By using B est’s method it proved possible to discover single granules of glycogen in this group of animals also.

G r o u p IV — the parathyroid glands of rats killed in the fourth and fifth w eek after rem oval of the parotid salivary glands.

The parathyroid glands of the animals in this group are also composed in the central part of dark cells with som ewhat elongated nuclei. The light cells lying peripherally, as in the control anim als, are arranged in clumps. These cells now have a slightly different appearance: they are high, almost cylindrical, large, and m ainly supported at their base by blood vessels. The nucleus is situated in the basic part of the cell (Fig. 15. Plate XIII). As was the case in group II, nuclei in course of preparation for division are abserved.

During this period a third kind of parathyroid elem ent appears — the oxyphil cells. These are cells with intensively acidiphilous cytoplasm and hyperchrom atic nuclei, situated in bands or clum ps (Fig. 16, P late XIII).

The Golgi apparatus in these cells is situated, sim ilarly to that in the control animals, on the apical pole. It is formed of single delicate trabeculae and small fragments arranged round the nucleus (Fig. 17, Plate XIII).

It would seem that this gland as a whole is enlarged compared to that in control animals. At the same tim e penetration is observed from the periphery of bands of connective tissue, which separate it from the sm aller and larger lobes.

3. R esu lts

1. The parathyroid glands of rats from which the salivary glands have been removed exhibit morphological and histochemical changes,

230 I. D zie rzyk ray -R o ga lsk a

2. The changes begin to occur in the second w eek after removal by operation of the salivary glands and affect the cellular nuclei in the first place. The follow ing are found:

a) presence of nuclei preparing for amitosisb) a large number of caryokinetic figuresc) increase in the number of large nucleid) characteristically large nucleoli situated in the im m ediate vicinity

of the nuclear membrane.3. The peripheral part of the gland formed of light cells undergoes

considerable growth.4. The light cells increase in size.5. O xyphil cells arranged in clumps appear.6. It would seem that the changes described above, and in particular

the presence of num erous cell divisions the increase in size of the nuclei and cells, constant arrangement of the nucleoli near the nuclear membrane and the increase in the number of light cells, m akes it possible to reach the conclusion that the parathyroid glands, after rem oval of the salivary glands, exhibit features which are evidence of the stim ulation of these glands.

VI. E F F E C T OF A D M IN IST E R IN G PA R O T IN ON T H E M O R PH O LO G IC A L

PIC T U R E O F T H E PA R A T H Y R O ID G LA N D S AND PA R O T ID SA LIV A R Y GLA ND S

The anim als in this group were given intramuscular injections of 0.1 mg. or 0.2 mg. of parotin per 100 g. body w eight during the course of 7— 14 days. A ll the rats w ere fed on a standard diet. D issection of the animalswas performed 24 hours after the final injection, and 12 hours after thelast feed.

During dissection specim ens were taken from the parotid salivary glands and the parathyroid glands. The material was fixed and stained by the methods described in Section III.

The anim als which received parotin w ere divided as follows:G r o u p I consisted of 5 control animals, which w ere given 0.2 mg.

saline solution per 100 g. body w eight, daily for 14 days.G r o u p II was composed of 10 anim als which w ere given 0.1 mg. of

parotin per 100 g. of body weight, daily for 7 days.G r o u p III consisted of 10 rats which were given 0.2 mg. of parotin

per 100 g. of body w eight, daily for 7 days.The f o u r t h g r o u p was formed by ten anim als which were given

0.2 mg. of parotin per 100 g. body daily for 14 days. A total of 35 animals were used for the experim ent.


A. P A R A T H Y R O ID G L A N D S


The picture of the parathyroid glands of rats which w ere given parotin, in groups II, III and also IV, is similar. The w hole of the gland appears to be formed of cells of one kind only, that is, from dark cells w ith dense and fine-grained cytoplasm , and as a result it is im possible to distinguish the peripheral part from the central part of the gland. The cellular nuclei give the im pression of being more hyperchrom atic. They differ in shape but are not large, and the nuclear m em brane is thick and often folded (Fig. 18, P late XIV). M orphologically these cells are sim ilar to the dark cells described above in the parathyroid glands of control animals, but they appear to be smaller. The w hole gland also appears to be smaller. H istochem ical investigations, to discover the presence of RNA acid, acid m ucopolysaccharides or PAS reaction did not give positive results.

The Golgi apparatus in the cells of the parathyroid glands is also situated in the apical part of the cells and has the appearance of a scattered formation (Fig. 19, P late XIV).

2. Results

1. The parathyroid glands of rats which w ere given parotin exhibit morphological changes consisting in the reduction in size of the nuclei, an increase in density of the cytoplasm atic granulations and reduction in the number of light cells.

2. The changes described do not vary with the dose and period of time during the parotin is given.

3. Changes occur in the structure of the gland as early as on the seventh day after the injection of parotin.

B. P A R O T ID SA L IV A R Y G L A N D S


The morphological pictures of the parotid salivary glands of rnts given parotin are identical in groups II, III and IV, but differ from the salivary glands of the control animals. In comparison w ith the control salivary glands, the nuclei of the cells forming vesicles alter their appearance in the animals in this group, and appear to be sm aller. They also change in shape, become variform and not infrequently a folded and tickened nuclear membrane can be observed. The sporadically encountered round

Í. D z ierźykray-R ogalskd

nuclei also have a thick and slackly stretched nuclear membrane. A fter parotin is adm inistered enlargem ent of the blood and lym phatic vessels situated near the striated ducts is observed to occur (Figs. 20, 21, PlatoXV).

In comparison w ith the salivary glands of the control animals, the argentophilous fibres w ith in the gland, which surround both the striated ducts and the blood and lym phatic vessels lying near them with numerous coils, increase in number.

After applying the Brachet reaction distinct reduction is observed of the amount of ribonuclein acid in the basic parts of the glandular cells (Fig. 22). The presence of P A S + granules is observed in the apical parts of the cells and also in the cells of the intercalar ducts (Fig. 23, P late XV).

W hen histochem ical m ethods were used the striated ducts gave no reaction.

2. Results

1. The parotid salivary glands of rats which were given parotin, exhibit both morphological and histochem ical changes occurring w ith uniform intensity, irrespective of the dose or period during which parotin is administered.

2. The changes observed occur in the gland as early as seven days after the first dose of parotin.

3. The above changes, reduction in size of the cellular nuclei, reduced pyronin absorption, increase in the amount of connective tissue) are evidence of the w eakened activity of the salivary glands of rats which have been given parotin.

V II. T H E E FF E C T O F REM OVAL OF TH E SA LIV A RY G LA N D S

AND A D M IN IST E R IN G PA R O T IN ON TH E A U T O R A D IO G R A PH IC P IC T U R E S

OF TH E BONE OF W H ITE RA TS

The experim ents described above show that the parathyroid glands intensify their activity as a result of the absence of parotid salivary glands. Parotin adm inistration causes morphological signs of functional inhibition both of the parathyroid glands and the parotid salivary glands. We were of the opinion that the morphological changes of the glands investigated should be reflected in the changes taking place in the skeletal system . On this account, several experim ents were made using radioactive P 32. 3)

3) T he w o rk w as c a rr ie d o u t in co llab o ra tio n w ith L. G u t s z e, M. Sc., in th e M ed ical Physics D e p a rtm e n t of th e M edical School, B iałystok .


1. D escrip tion of m a te ria l

A total of 30 young w hite rats kept on a standard diet w ere used for the investigations,, and w ere divided into the follow ing 6 groups:

'Group I — control animals.Group II — anim als from which the parotid salivary glands had been

removed, and dissection made a w eek after the operation.Group III — anim als which w ere dessected 2 weeks after the removal

of the salivary glands.Group IV — anim als which w ere dissected 3 w eeks after rem oval of the

salivary glands.Group V — anim als which w ere given 0.1 mg. of parotin per 100 g.

body w eight, daily for 7 days.Group VI — anim als which w ere given 0.2 mg. of parotin per 100 g. of

body w eight daily for 7 days.

imp/m in.

Fig. 24. D iag ram of th e ac tiv ity of bone ash ca lc u la ted by m ean s of a G eig er-M tillc r co u n te r .

1 — C on tro l. 2 — 7 days a f te r rem o v in g of sa liv a ry g lands. 3 — 14 d ay s a f te r rem o v in g of s a liv a ry g lands. 4 — 21 days a f te r rem o v in g of sa liv a ry g lands . 5 — A fte r

in jec tio n s 0.1 m g. of p a ro tin . 6 — A fte r in jec tio n s 0.2 m g. of p a ro tin .

In order to trace the behaviour of phosphorus in the bones, radio-active phosphorus was used in the experim ent, injecting 0.08 m icro-Curies P*2 (Na2H32P 0 4 — alkaline sodium phosphate) per 1 g. of body w eight. The tibiae of each anim al w ere prepared, one of which (the right) was burnt in a m uffle stove at a tem perature of 900°C., and the num ber of im pulses calculated from the ash obtained by m eans of a G eiger-M iiller counter. The results obtained are shown on Fig. 24.

M icro-sections w ere made from the epiphysis of the second bone (left t ibia) in order to obtain autoradiograms, which w ere made by the Pelc method using AR-10 Kodak em ulsion. A fter developing the picture on the em ulsion photographs was made by the contact m ethod. The results

TU i. D ziedykray-Rogalska

obtained from the above expeiiment are shown on Figs. 25— 30 (PlateXVI).

The largest num ber of impulses in the bone ash were obtained in Group VI of the anim als (which were given 0.2 mg. of parotin for 7 days). The autoradiographic picture of the base of the bones in this group of animals exhibits the greatest degree of blackening of the film.

The sm allest number of impulses from bone ash were obtained in group II, i.e. of the anim als from whica the parotid glands had been removed. These results w ere confirmed by the autoradiographic picture, which ex hibits very faint blackening of the film in the bases of the bones in this group of animals.

2. Results

1. The greatest activity in bone ash was obtained in the animals from group VI (which w ere given 0.2 mg. of parotin), and the autoradiographic picture corresponds to this.

2. The sm allest number of impulses were obtained in bone dust in group II of the anim als (animals killed one week after removal of the salivary glands), and the autoradiographic picture corresponds to this.

V III. ST A T IST IC A L A NA LY SIS OF M A TERIA L

Evaluation of the activity of the parythyroid glands based on morphological methods is difficult, and therefore statistical methods were used in the expectation of obtaining more far-reaching results, which m ight confirm or extend the morphological observations described above.4)

As show n in the descriptions contained in the preceding sections, the parathyroid glands of animals from which the parotid salivary glands had been removed, are in a stimulated condition. The administering of parotin caused changes in the parathyroid glands constituting evidence of the inhibition of their activity. As is w ell known, one of the phenomena occurring w ith increased activity of the ductless glands is the observed enlargem ent of the nuclei. Reduction of activity, on the other hand, is characterised by the distinct reduction in the size of the nuclei.

W e were interested in whether a statistical analysis based on data obtained from counting the nuclei occurring in a permanent field of vision w ould confirm the changes in size of the nuclei in the parathyroid glands of anim als in d ifferent experim ental groups.

4) T he s ta tis tic a l ana ly s is of th e m a te r ia l w as m ade in th e In s titu te of S ta tis tic a l M ath em atic s of th e M aria C u rie -S k lodow ska U n ivers ity in L ub lin , u n d e r th e d ir e c tion of P ro f. D r. M iko la j O l e k i e w i e z .

Table 1.

M ean n u m b e r of n uc le i fo r each ra t.

Control /£/ Removal of salivary glands /U/ Parotyna a /No.of

rat

Periphefiral part of gland, eld of vision

Central part of gland

No.ofrat

Peripheral part of gland, field of vision

Central part of Klaftd

No.ofrat

Peripheral part of gland, field of vision

Central part ofgXotrfcd1st 2nd TotaJ 1st 2nd Total 1 at Pn4 Total

23 80. 00 91.00 85.50 89.00 59 08.00 72. 07 70. 33 63. 33 124 75. 67 80.33 78. 00 78.6724 83.00 92.33 87. 67 71.67 52 80.33 77. 00 78.6’ 72. 00 125 73.67 83.67 78.67 77. 0049 52.33 57.00 54.67 56.33 112 74.33 81. 83 77.83 72.67 127 76.33 80.33 78.33 82.6745 70. 00 77.33 73.67 71.33 51 77.67 71. CO 74.33 65. 00 e5 74.33 81.67 78.00 82. 00

114 89.67 103.00 96.33 84.00 62 64.00 63. 00 63.50 64.33 83 88.67 68.33 78. 50 77.0048 66.00 70. 00 68. 00 73.00 60 63.00 58.67 60.83 55.67 81 74. 33 80. 00 77.17 76.67

Total 73.50 81.78 77.64 74.22 Total 7 1 . 2 2 70. 61 70.92 65. 50 Total . 77.17 79.06 78.11 79.00

23b 1. D zic rzyk ray -R og a lska

M a te ria l w as co llected consisting of 18 ra ts , 6 in each of the. g roups ex am in ed , as fo llow s:

G ro u p I — c o n tro l (K),G ro u p II — an im als fro m w hich th e p a ro tid g lands had been rem oved (U),G roup I I I — an im als w h ich w ere g iven in jec tio n s of p a ro tin (Z ).T h ree d if fe re n t m ic ro -sec tio n s w ere ta k e n from each p a ra th y ro id g lan d of o b

se rv a tio n . C a lcu la tio n w as m ad e of th e n u m b e r of c e llu la r nucle i in th re e fie lds of v is ion on each sec tion , m easu rem en ts a lw ays be in g m ad e of tw o fie lds ly ing opposite each o th e r on th e p e rip h e ry of th e sec tion and of one fie ld in th e c e n tre of th e se c tion. In th is w ay m e a su re m e n ts of th e n u m b er of nucle i in n in e fie lds of v is ion w ere o b ta in ed fro m one p a ra th y ro id g land . In o rd e r to co u n t th e n u m b e r of nucle i in a co n s tan t fie ld of v is ion a m ic ro m etric o b jec tiv e m esh w as p laced in 15 X m ag n ify ing ocu lar, th u s o b ta in in g a sq u a red fie ld w ith 11 h o rizo n ta l and 11 v e rtic a l lines. T he p re p a ra tio n s w ere observed w ith an im m ersio n o b jec tive o b ta in in g 1500 < m agn ifica tion .

T he n u m b e r of c e llu la r n u c le i o ccu rrin g on 11 h o rizo n ta l and 11 v e r tic a l lines w e re coun ted in each fie ld of v ision . In ad d itio n th e nucle i occu rrin g on th e p o in ts a t w h ich th e tw o lines crossed, w ere coun ted , th e to ta l n u m b e r of n u c le i occu rrin g on th e crossing w e re d ed u c te d fro m th e sum of all th e n u c le i p re s e n t in th e g iven fie ld of v ision , o b ta in in g in th is w ay th e t ru e sum to ta l of n u c le i o ccu rr in g in one £ ield of v ision .

T he f i r s t ta sk w as to ca lc u la te th e m ean n u m b ers of th e nucle i fo r each an im al of th e th re e g roups.

S ince it w ould seem fro m m orpho log ica l o b se rv a tio n s th a t th e cells ly ing in th e p e rip h e ry of th e g lan d re a c t fa r m o re qu ick ly to th e s tim u li by a change in th e ir size and a p p ea ran ce th a n th e cells ly ing in th e c e n tra l p a r ts of th e g land , th e m ean n u m b ers of nu c le i fo r each of th e an im als w ere ca lcu la ted se p a ra te ly fo r th e c e n tra l and p e r ip h e ra l fie ld s of v ision , using in th e f i r s t case 6 fie lds of v ision , and in th e second , 3 fie lds of v ision . T hese m ean n u m b ers of n uc le i fo r each r a t a re g iven in ta b le 1. In ad d itio n , in th e b o ttom line of ta b le 1 th e m ean n u m b ers of nucle i in th e p e rip h e ry and c e n tre of th e sec tions a re g iven fo r each of th e th re e g roups of a n im als (T able 1).

T he re la tio n s show n in ta b le 1 a re i l lu s tra te d by d iag ram (Fig. 31) re p re se n tin g th e d is tr ib u to n s of th e se r ie s of th e m ean n u m b ers of nuclei. E ach p o in t re p re se n ts a se p e ra te an im al, and th e position of th is p o in t h ig h e r o r low er, th e p lace of th e an im a l in its g roup .

Parotin acts on the parathyroid gland, prim arily in the peripheral parts of the gland (Fig. 31). In the group of anim als given parotin the mean of nuclei of the parathyroid cells exhibit hardly any variation, concentrating strictly around one figure — 78, w hile in the control group the dispersion of the mean numbers of nuclei is very considerable in relation to the concentration described. This m eans that the doses of parotin act in two directions — in the anim als w ith mean numbers of nuclei greater than 78 this factor reduces their averages to the figure of 78. In anim als w ith mean numbers of nuclei sm aller than 78, this factor increases their averages to the figure 78. This conclusion is further borne out by the variations found in the m ean numbers of nuclei in the control group (in this group half the animals had mean values far above 78, and half far below


78). It may therefore be taken that the group of animals injected w ith parotin would have exhibited a dispersion of averages similar to the control group, if they had not been given parotin.

In the central parts of the sections (Fig. 31) a similar effect of parotin is found, but not to the same degree as in the periphery. Analogical concentration round the number 78 occurs in four animals, the two remaining anim als concentrating round 82. It m ay be assumed here that

Peripheral part Central partof gland of gland

Fig. 31. M ean n u m b e r of c e llu la r nucle i in th e p a ra th y ro id g lands of ra ts .1 — C ontrol. 2 — R a ts w ith sa liv a ry g lands rem oved . 3 — R ats w ith in jec tions of

p a ro tin .

the effect of parotin on the central parts of the gland is weaker than on the peripheral parts, particularly where one of the two directions of action is concerned, the reduction of the number of nuclei in anim als with high mean values.

Assum ing that the greater the number of nuclei observed in the field of vision, the sm aller their dim ensions, and vice versa (with a constant ratio of nuclei-cytoplasm ) it may be stated that in the parathyroid glands

23« I. D zic rzyk ray -R o ga lsk a

w ith, on an average, large nuclei, that parotin acts in the direction of reducing their dimensions. In parathyroid glands w ith on an average sm all nuclei parotin induces their enlargem ent. It must therefore be presumed that the first action (reducing) is stronger than the second (enlarging). W hat is very striking is that the effect of parotin on the periphery tends to equalise the mean dim ensions of cellular nuclei in the parathyroid glands of different anim als to a certain dimension corresponding to the number of 78 nuclei in a field of vision.

The effect of excising the salivary glands is also interesting. This effect, as can be seen from diagram (Fig. 31), is exerted, it is true, in one direction, but it is exerted selectively. This influence consists in the enlargem ent of the mean sizes of the nuclei, but rather in those animals only in which these mean values are small (i.e. mean number of nuclei is large). This influence is apparent both in the periphery and in the central parts af the gland.

The question arises as to whether the two-direction action, or selectivity, of the influences of experim ental factors on the mean dim ensions of the nuclei exists w ith in the peripheral or central parts of each gland as w ell as betw een glands. Within the scope of action of one factor total variability (either in the periphery or in the centre of the glands) consists of variability betw een animals and of the mean variability w ithin the anim als, and the latter in the mean variability betw een sections and the m ean variability w ithin the sections. The ’’mean squares” in the hierarchic variance analysis (F e d e r e r, 1955) are the com parative m easures of these different variabilities. In the term inology used in this analysis the questions put would read: do the experim ental factors reducing the mean squares betw een animals also reduce the m ean squares w ithin the animals,i.e. the mean squares between the sections and the mean squares w ithin the sections? The answer to this question is given by table 2, which contains the results of three hierarchic variance analyses made separately for the control group and experim ental groups. Table 2 shows the fo llow ing facts:

1° The dominating component of total variability in control group (K ) and in the group of rats with their salivary glands removed (U) is formed by the differences betw een rats, particularly in the peripheral parts of the gland, e.g.: in group (K) the mean square betw een rats (1378) is nearly 13 tim es greater than the mean square w ith in the rats (110).

2° In the peripheral parts of the glands in group (K) and in group (U) the differences betw een sections do not create an additional variation which could not have resulted from the variation w ithin the sections, since, for exam ple, in group (K) the mean square betw een sections (124) is only slightly greater than the mean square w ithin the sections (101). It

T ab le 2.

H ie ra rch ic an a ly s is of variance .

Number Control /K/ Removal of salivary gland /U/ Parotyna / 2 /Source of variation of degree

of freedomSum of squares

Meansquare

Sum of squares

Meansquare

Sun of squares

Meansquare

A. Peripheral part of gland ; 6 animals x 1 micro-sections x 2 f eld of visicnbetween rats 5 6. S89. 50 1. 377. 90 1. 659. 58 131.92 8. 56 1. 71within rats 10 1. 29A. 81 109. 83 1.865.i7 62.17 2.879.00 95.97

betweenmicro-sections

In particularwithiAmicro-sect ions

12

18

1.AS3.11

1.611. 50

123. 61

ICO. 6A

737.67

1.127.50

61.47

62. 64

62A.00

2.255.00

52. CO

125.28

T o t a l 15 10. 18a . 11 - 3. 52A.75 - 2.887. 56 -

B. Central part of gland 6 animals 3 micro-sections x 1 field of vision

between rats 5 1.951.11 190.22 589.03 117.97 1 08. 00 21.60within rats /between micro-sections/ 12 1. 88a. 00 i 57. 00 182.67 11.89 8A8.00 70.67

T o t a l 17 1.835. 11 - 972. 50 - 956.00 -

239

240 I. D zie rżykray -R ogalska

may therefore be said that there are no significant differences between the sections.

3° In the group given parotin (Z) differences in the mean number of nuclei betw een the rats disappear, falling far below their random variability which would have resulted from the variability w ithin the glands. This phenom enon occurs particularly distinctly in the periphery of the gland, w here the m ean square betw een the rats falls to the figure 2, w hile the mean square within the rats is 96. The difference between micro-sections also falls below random variation which would have resulted from variations w ithin the sections, but to a far lesser and non- -significant degree than the differences betw een animals. These facts give evidence of the intensive equalising action of parotin.

Table 3.

C om parison of m ean sq u ares in th e ex p erim en ta l g roups and in th e co n tro l group .

Comparison or mean squaresComparison of the control grup /F/

F0.05Humber of̂ degree of freedom

with the group with salivary glands removed /0/

with the group given injections of parotin /2/

A. Peripheral p;<rt of ¿landbetween rats within rats

4.1 5 1.77

804.851.14

5.05i.ei

5 i 5 30 ; 30

betweenmicro-sections

In particulaj withinmicro-sections

P . 0 1

1. 6 1

i

CO!

'""V CsJ

■NJ V-

2. 69

2.22

12 ; 12

18 ; ię

Q. Central part of glandbetween rats within rats/between nicro-sections/

3. 31

4 . 92

16. 07

2.22

5.05

2. 69

5 ; 5

1? ; 12

Table 3 gives the results of comparison of the experim ental groups with the control group from the aspect of:

1) variations betw een animals and 2) variations within the animals.In the peripheral parts of the glands variation w ithin the anim als was

divided into variations:a) betw een m icro-sections, and b) within the micro-sections.In the central part of the glands the variations betw een micro-sections

correspond to the variations within the animals.In each case comparison was made of the mean squares in groups U and

Z w ith the mean squares in group K ( F i s h e r , 1957).G enerally speaking, the mean square w ithin the rats was reduced

significantly only in group U, being reduced to a far greater extent in the

P a ro t id sa l iv a ry g lands and pa ra th y ro id g lands 241

central parts of the glands than in the peripheral parts. In group Z this reduction, despite the fact that it is not significant, is also greater in the central part of the glands.

The equalising action of parotin was most distinctly marked, particularly in the peripheral parts of the glands. Removal of the salivary glands had a far lesser influence, and this influence m anifested its equalising action in a different way.

The m axim um equalising action of parotin wTas evident in the d ifferences betw een animals. These differences were in fact elim inated, and also the differences betw een micro-sections were reduced, even if to a considerably smaller, still to highly significant degree. The differences within the m icro-sections were rather increased by parotin (although not- significantly) so that the differences as a whole w ithin the animals remained unchanged in comparison with the control group.

The equalising effect of removal of the salivary glands on the d ifferences betw een the animals was slightly greater in the periphery in comparison w ith the central part of the glands. Although this difference cannot be considered significant (4.15 and 3.31 — from table 3), it is a phenom enon analogous to the stronger effect of parotin on the periphery than on the central parts of the glands.

If on the other hand w e put a question of the equalising effect of the experim ental factor on the differences between m icro-sections, it is particularly here that this effect proved to be most marked, but only in the central parts of the glands, where it is even more marked than on the differences betw een animals, which is a converse phenomenon to that in the case of parotin.

Thus w hile the equalising action of parotin primarily affects the d ifferences betw een animals and is far stronger in the periphery than in the central part of the glands, the effect, generally far weaker, of removal of the salivary glands primarily equalises the differences betw een micro- -sections and rather in the central parts of the glands.

One important fact remains to be emphasised, that is the absence of significant differences betw een micro-sections in all three groups (Tab. 2). This fact gives evidence of the great similarity of structure of m icro-sections from different places in the same gland, although the m icro-sections have d ifferent properties in the periphery and in the centre of the gland. This is of great importance to the research worker, since it gives rise to the assumption that regardless of the part of the gland from w hich the m icro-secticn is taken, this micro-section is fu lly representative of the structure of the whole gland in the given animal.

This assumption should of course be additionally checked by taking sections from different defined places in the gland.

242 I. D z ierzykray-R ogalska

On the other hand differences betw een anim als, i.e. d ifferences between the structures of d ifferent glands, are very large.

If the absence of significant differences betw een the micro-sections w ere confirmed by special experim entation, it would then appear that the most correct procedure in research on the parathyroid gland is to take the greatest number of animals and lim it the number of m icro-sections taken to tw o only, or even to one micro-section from each anim al — of course taking into consideration separately the peripheral and central parts of the m icro-sections.

IX . C O N SID ER A TIO N OF R ESU LTS AND D ISC U SSIO N

This study was intended to supply an answer to the question as to w hether there is a connection betw een the parathyroid glands and the parotid salivary glands — which are connected w ith the calcium -phos-

Table 4.

P lan show ing re su lts of in v es tig a tio n s.

Parotoid salivary glands

Parathyrold glands

Activity of bone ash and autoradiographic picture of the bases of bones32after administering P

Removed of parathyroid glandsRemoval of parotid salivary glands

—

+ .

Injection of paratin

— — +

phorus m etabolism of the organism. With this aim in view a series of experim ents w ere made.

The results of the investigations have been set out in plan form in table 4.

As shown by the experim ents made, the absence of the parathyroid glands causes considerable morphological changes in the parotid salivary glands of w hite rats. These changes occur in the secretory vesicles, the lum en of w hich at first enlarges considerably, and later becomes filled w ith finegrained secretion (Fig. 4). Vacuoles appear in the vesicular cells, then the cells flatten. At the same tim e the connective tissue, w ith a large amount of fatty cells, penetrates betw een the changed vesicles of the gland. As the process continues, only fatty tissue is observed in the place of the lobes w ith altered structure, and numerous striated ducts can be seen in the fatty tissue as the residue of the glandular lobes (Figs. 8, 9).

Paro t id sa l iv a ry g lands an d p a ra th y ro id g lands 243

The morphological pictures described above lead to liquidation of part of the glandular lobes, and in consequence to partial withdrawal of the salivary glands from their function.

The view s held by different authors on the structure of the parathyroid glands do not agree. It is true that there is a certain coincidence of view s on the morphological classification of the glandular cells, but the fact that in different mammals slightly different types of cells occur, complicates the situation.

The majority of authors sonsider that no oxyphil cells are observed in the parathyroid glands of lower mammals in their normal state. These cells are only found in certain pathological conditions induced by experiment. F o r t i n a (1948), for instance, noticed the appearance of cells with acidophilous cytoplasm in the parathyroid glands of rats after rachitis had been induced in these animals. C a s t l e m a n & M a l l o r y (1935), B a r g m a n n (1939), C o r r e i a M a d e i r a (1942) hold the view that oxyphil cells are degenerative elem ents formed from the chief cells. This view is supported by H a m p e r l (1950), who stated that oxyphil cells are formed by a special kind of degeneration to which the cells of other glands are also subject. Analogical cells filled with eosinophilous granulations (oncocytes) are found in the thyroid, pituitary and suprarenal glands.

A description has been given in the present investigations of the presence of two kinds of cells (dark and light) in the parathyroid glands of the control rats, and the additional appearance of cells w ith acidiphilous cytoplasm in the glands of anim als from which the parotid salivary glands had been removed (similar cells were observed by E d e r, 1961). We cannot give an opinion as to the origin of the cellular forms described, since this still remains a debatable question, despite the application to research on this problem of both electronic microscopes and sensitive histochem ical and enzym atic investigations. It would, however, seem that all these cells develop from one type of cell, and the differences in appearance depend on a different functional state. The histochem ical methods w e used did not assist in the solution of this problem, but it would appear that the increase observed in the number of light cells — considered as active cells (in the peripheral parts of the glands) and of oxyphil cells, the presence of a large number of nuclei preparing for amitosis and frequent m itoses (Fig. 12), and also the position of the nucleolus near the nuclear membrane, form sufficient grounds for concluding that the parathyroid gland goes through a period of intensified activity after the rem oval by operation of the parotid salivary glands.

A statistical analysis made it possible in addition to establish dispersion of the mean numbers of nuclei in the parathyroid glands of rats w ith their

244 I. D z ie rzy k ray -R og a lska

salivary glands removed, and at the sam e tim e indicated the significance of the deflection reducing the m ean number of the nuclei referred to. This leads to the conclusion that larger nuclei occur in the parathyroid glands of this group of animals in comparison w ith the control group (Fig. 31).

The morphological pictures of the parathyroid glands of animals given parotin differ only slightly betw een them selves, but reduction of the part of the cellu lar nuclei observed, and also the reduction of the cells perm its the assum ption that these glands were subject to reduction as compared w ith the glands of the control rats. The morphological pictures described would therefore seem to show that the parathyroid glands, after parotin is adm inistered, exhibit a d istinct w ithdrawal from activity.

A statistical analysis showed that the effect of parotin is to render uniform the m ean dim ensions of the nuclei, causing as a result reduction in size of the large nuclei, but at the sam e tim e causing the enlargem ent of the sm allest nuclei (Fig. 31).

The m orphological pictures of the parotid salivary glands after inject- ̂ions of parotin also exhibit m orphological features consisting in the reduction of the term inal sections and their nuclei. At the sam e tim e the disappearance of RNA from the basic parts of the vesicular cells can be observed, which gives grounds for concluding that the secretory activity of the gland is reduced.

A decrease in the activity of P32 was observed in the bone ash from rats from which the parotid salivary glands had been removed.

An increase in radioactive phosphorus was found in the anim als which were given parotin. The autoradiographic pictures correspond in both groups to the num erical results.

As show n by the experim ents made (Sections V and VI) supported by the results obtained from the statistical analysis, after removal of the parotid salivary glands stim ulation of the activity of the parathyroid glands is observed. The injection of parotin, on the other hand, causes inhibition of the activity of the parathyroid gland.

The reply to the question w hy removal of the salivary glands causes hyperfunction of the parathyroid glands must be based on the process ;if reciprocal action of both glands. It must be accepted that the parathyroid gland is the m aster gland (producer) in relation to the parotid salivary gland. A fter rem oving the parotid salivary glands that is, the effector, the intensified activity of the parathyroid gland observed becomes understandable, and conversely, injection of parotin, that is, the induction by experim ental m eans of a state of hyperfunction of the parotid salivary glands (the effector) caused blocking of the function of the parathyroid glands (the producer).

Assum ing that the parathyroid glands are the master glands both in the

P aro t id sa l iv a ry g lands a nd p a ra th y ro id glands 245

calcium-phosphorus m etabolism of the bones (tissue effectors) and in relation to the parotid glands, the results described in Section IV become understandable. After rem oving the parathyroid glands (the producers) it was found that morphological changes take place in the parotid salivary glands indicating the atrophy of the glandular tissue, w hich m ay be evidence of the withdrawal of a certain part of the glandular parenchyma from functioning.

An attem pt can be made to explain the functional connection of these tw o glands by referring to the pituitary gland. The m ajority of the authors dealing w ith the dependence of the parathyroid gland on the pituitary gland reach the conclusion, however, that the parathyroid glands possess far-reaching functional independence, based more on variations in the mineral m etabolism than on connection w ith the pituitary gland. In analysing the role of the parathyroid glands in the hormonal system in respect to variations in m ineral econom y the conclusion m ust be reached that there is no direct connection betw een the parathyroid glands and other endocrine glands.

Connection of the endocrine function of the glands exam ined is more d ifficult since the existence of the parathyrotropic horm one remains a debatable question up to the present time. Parotin is also a hormone w ith m ulti-lateral action and there is no adequate proof that the endocrine function of the salivary glands is subject to the direct influence of the pituitary gland. Connection of the changes observed to take place in both the glands described through the pituitary gland is therefore som ewhat doubtful.

As a result of removal of the parotid salivary glands a reduced level of P 32 is observed in the bone tissue. Injection of parotin, on the other hand, causes considerable increase of radioactive phosphorus, as was established both by means of a Geiger-M iiller counter and autoradic- graphically.

A very interesting problem is to find w hether the parotid salivary glands act directly on the bone tissue or through the m edium of the parathyroid glands.

Rem oval of the salivary glands causes on the one hand hyperfunction of the parathyroid glands (Section V) and on the other reduction of the level of P 32 in the bone (Section VII). Injection of parotin inhibits the function of the parathyroid gland and at the sam e tim e influences the increased P 32 content.

The question therefore arises as to what is the m echanism causing the changes described above.

The facts observed and described above allow m e to offer two hypotheses. The first of these would indicate the direct influence of the

246 I. D zie rzyk ray -R o ga lsk a

parotid salivary glands on bone tissue, w hile the second possibility would refer to the inclusion in this mechanism, additionally, of the parathyroid glands.

The direct influence of the parotid glands on the bone tissue is shown in the studies by Japanese authors ( S a s a k i et al., 1953; O g a t a , 1955; 11 o et al., 1956). By injecting parotin — the hormone of the parotid salivary glands, distinct stim ulation was obtained of the tissues of mesen- chym atous origin and calcification of the hard tissues.

If, however, the parotid salivary glands are removed, a very characteristic reduction in the level of P 32 is observed in the bone.

Fig. 32. F u n c tio n a l connections be tw een th e p a ra th y ro id g lands, p a ro tid sa liv a ryglands and bone tissue .

These facts em phasise the direct connection of the process of calcification of the bones w ith the parotid salivary glands.

At the present tim e there are no indisputable proofs of the direct action of the parotid salivary glands on the process of calcification of the bones, but the finding of changes in the functioning of the parathyroid glands both after rem oval of the parotid salivary glands and after giving parotin argues, on the basis of the function of the parathyroid glands known in physiology, in favour of the possibility of stim ulation of the processes of calcification of the bones by the parotid salivary glands.

P aro t id sa l iva ry g lands and p a ra th y ro id g lands 247

The results obtained by the experim ents concerning the changes in the parathyroid glands and parotid salivary glands indicate a functional interdependence betw een these two glands.

It is d ifficult on the basis of the facts given to form an opinion as to the role and hierarchic dependence of the parotid salivary glands and parathyroid glands in the calcium-phosphorus metabolism. The existence of the superiority of the parathyroid glands in the function discussed may how ever be suggested, the role of the salivary glands being then lim ited to that of a link in the chain of essential connections in m ineral m etabolism. At the same tim e this would be the role of organic effector of the parathyroid glands, the hypofunction or absence of which would lead to very significant damage to the calcium-phosphorus m etabolism.

The functional connections betw een the parathyroid glands, the parotid salivary glands and bone tissue are illustrated by Fig. 32.

X. SU M M A RY

T he a u th o r s tu d ied th e connection b e tw een th e p a ra th y ro id g lands and the p a ro tid sa liv a ry g lands — g lands connected w ith th e ca lc iu m -p h o sp h o ru s m etabo lism of th e o rgan ism . F o r th is p u rp o se she in v es tig a ted :

1. T he e ffec t of rem oval of th e p a ra th y ro id g lands on th e m orpho log ica l p ic tu re of th e p a ro tid sa liv a ry g lands.

2. T he effec t of rem o v a l of th e p a ro tid sa liv a ry g lands on th e m orpho log ica l p ic tu re of th e p a ra th y ro id g lands.

3. T h e e ffec t of in jec tin g p a ro tin on th e m orpho log ica l p ic tu re of th e p a ra th y ro id g lands and p a ro tid sa liv a ry g lands.

4. T he e ffec t of rem o v a l of th e sa liv a ry g lands and in jec tio n of p a ro tin on th e bones of ra ts . T he in v es tig a tio n s w ere m ad e using P 32.

5. In add ition a s ta tis tic a l ana ly s is w as m ade of th e m a te r ia l ex am ined .A fte r rem o v a l of th e p a ra th y ro id g lands a tro p h y of th e g la n d u la r tis su e occurs

in fa v o u r of fa t ty tissue , w h ich m ay be ev idence of th e w ith d ra w a l of c e rta in p a rts of th e g la n d u la r p a ren ch y m a from function .

As a re s u lt of excis ing th e sa liv a ry g lands, th e fo llow ing changes w ere caused in th e p a ra th y ro id g lands: e n la rg e m e n t of th e d im ensions of th e g la n d u la r cells and th e nucle i, th e p resence of n u m ero u s cell d iv isions, p e rm a n e n t position of th e nucleo lus n e a r th e n u c lea r m e m b ra n e and in c rease in th e n u m b er of lig h t cells — the ac tive cells. T he above d a ta in d ica te th a t th e p a ra th y ro id g lan d s e x h ib it sym ptom s of s t im u la tio n a f te r th e sa liv a ry g lands h av e been rem oved .

A fte r c a rry in g o u t ex p e rim en ts consisting in ad m in is te rin g p a ro tin th e fo llow ing re su lts w e re o b ta in ed : red u c tio n of th e nucle i, red u c tio n of th e p y ro n in ab so rp tio n of th e p ro to p lasm , in c rease in th e am o u n t of connec tive tissu e in th e p a ra th y ro id g lands and dec rease of th e cells and nucle i in th e vesicles of th e sa liv a ry g lands. T hese o b se rv a tio n s a re p roof of th e en feeb led ac tiv ity bo th of th e p a ra th y ro id g lan d s and of th e sa liv a ry g lands in ra ts g iv en p a ro tin .

R a ts f ro m w hich th e s a liv a ry g lands h ad been excised an d ra ts w h ich h ad been g iven p a ro tin w ere used fo r th e au to rad io g rap h ic in v estig a tio n s. U sing rad io ac tiv e

I. D z ie rzykray-R ogaiska

P 32, ex am in a tio n w as m ad e of th e in fluence of th e above fac to rs on th e p hospho rus level in th e bones of th e ra ts . The g re a te s t a c tiv ity in th e bone ash w as o b ta in ed in an im als w h ich w e re g iv en 0.2 mg. of p a ro tin p e r 100 g. body w eig h t over th e course of one w eek . T he au to rad io g rap h ic p ic tu re ag rees w ith th ese re su lts . T h e sm a lle s t n u m b e r of im pulses w as found in th e bone ash of an im als w h ich w ere d issec ted one w eek a f te r rem o v a l of th e sa liv a ry g lands, w h ich co rresponds to th e a u to rad io g rap h ic p ic tu re .

T he s ta tis tic a l a n a ly s is m ade of th e m a te r ia l en ab led th e fo llow ing to be estab lish ed :

a) la rg e r nucle i occur in th e p a ra th y ro id g lands of an im als from w hich th e sa liv a ry g lands h av e been rem oved in com parison w ith th e con tro l an im als .

b) n e ith e r very la rg e n o r very sm all nucle i are en co u n te red in th e p a ra th y ro id g lands of an im a ls as th e re s u lt of th e p a ro tin in jec tions g iven, i t w ould seem th a t th e p a ro tin e x e rts an eq u a lis in g effect, re n d e r in g th e m ean d im ensions of th e nucle i u n ifo rm .

As can be seen fro m th e above data , rem o v a l of th e p a ra th y ro id g lands caused m orpho log ica l changes in th e pa ro tid sa liv a ry g lands w hich p rov ided ev idence cf th e w ith d ra w a l of th ese g lands from th e function ing .

On th e o th e r h a n d rem o v a l of th e sa liv a ry g lands caused h y p e rfu n c tio n of the p a ra th y ro id g lands, an d in jec tions of p a ro tin caused b lock ing of th e fu n c tio n of th e p a ra th y ro id g lands.

T he above re su lts w ou ld ap p ea r to in d ica te th e ex is ten ce of re c ip ro ca l ac tion of bo th g lands, th e p a ra th y ro id g land being th e m as te r g land (producer) in re la tio n to th e sa liv a ry g lan d (effector).

I t is also c lea r fro m th e exp erim en ts m ade th a t th e sa liv a ry g lands e x e r t th e ir in flu en ce n o t only on th e p a ra th y ro id g lands, b u t also on the bone tissue . R em oval of th e p a ro tid s a liv a ry g lands causes ch a rac te ris tic red u c tio n of th e P 32 leve l in th e bone, w h ile in jec tio n s of p a ro tin causes an increase in P 32 con ten t. On th e basis of th e re su lts o b ta in ed th e a u th o r pu ts fo rw a rd tw o h y p o theses as to th e in flu en ce of th e sa liv a ry g ian d s on bones. A ccording to th e f ir s t of th ese th e p a ro tid sa liv a ry g land d irec tly affec ts th e bone tissue, accord ing to th e second — it does so th ro u g h th e p a ra th y ro id g lands, w hich, as is a lready know n, th en e x e r t a decisive in flu en ce on p h o sp h o ru s-ca lc iu m m etabo lism .

A ck n o w led g em en ts: I t is m y p leasan t d u ty to th an k D ocen t D r. H . L e w i n s k a , H ead of th e D ept, of H isto logy and E m briology, fo r th e ass istance and c ritic ism d u rin g th e p re p a ra tio n of th is pub lication .

I shou ld also lik e to th a n k P ro fesso r D r. M. O l e k i e w i c z fo r his h e lp in w o rk on th e s ta tis tic a l analy sis. My sincere th a n k s are due to D r. K. O d y n i e c fo r c a rry in g o u t th e m icro p h o to g rap h y , and Miss G. S t a c h o w s k a , fo r h e r tech n ica l assistance .

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87. M o r g a n , J . R. E., 1936: T he p a ra th y ro id g lands. A rch . P a th ., 21: 10—26.88. M y a n t, N. B., 1960: Iod ine m etab o lism of sa liv a ry g lands. A nn. N ew Y ork

A cad. Sc., 85: 208— 214.89. O g a t a , T., 1955: T he in te rn a l sec re tio n of sa liv a ry g land . E ndocr. Jap ., 11:

1— 15.90. O 1 e k i e w i c z, M.: 1956: S ta ty s ty k a jak o m e to d a poznaw cza. Z eszy ty p ro b le -

m ow e „K osm osu” , 2: 102—235. W arszaw a.


91. O s t r o w s k i , K., 1957: M ik ro au to rad io g ra fia . M etody, zasto so w an ie i w y n ik i. F o lia m orpho l., 8, 16: 128.

92. P a r h o n , C. I., B a b e s , A., P e t r e a, I. & B u r g h e r , E., 1955a: T e sticu lu l la sobo lan ii a lb i p a ro tid ec to m iza ti u n i si b ila te ra l. S tu d ii si c e rc e ta r i de E ndocr., 6, 3—4.

93. P a r h o n , C. 1., B a b e s , A., P e t r e a, I. & I s t r a t i, F., 1955b: A su p ra s tru c - tu r i g lan d e lo r p a ro tid e de sobo lan in ra p o r t eu v ir s ta si m o m e n tu l a p a r itie i d im o fism u lu i sex u a l. C om un icare A cadem iei R. P. R., 5: 12, 1—310.

94. P a r h o n , I. C., P e t r e a, I. & S a p o s n i c, Al., 1956: A su p ra a c tiu n ii h ip e r - g licem ian te a u n u i e x tr a c t de p a ro tid a . S tu d ii si c e rc e ta r i de E ndocr., 7: 2.

95. P a r h o n , I. C., B a b e s , A. & P e t r e a, I., 1957: E n d ocrino log ia g la n d e la r s a liv a ry . A cadem ia R epub lic ii P o p u la re R om ine. B ib lio teca M édicale , 5: 1—200.

96. P e a r s e, A. G. E., 1957: H istochem ia teo re ty czn a i s to so w an a . P a ń s tw . Z ak ł. W yd. L ek.: 1—535. W arszaw a.

97. P e a r s e, A. G. E. & T r e m b l a y , G., 1958: L euc ine am in o p ep tid a se in r a t p a ra th y ro id and its re la tio n to p a ra th y ro id ho rm o n p ro d u c tio n . N a tu re , 181: 1532— 1533. L ondon.

98. P i c a r d , J., 1955: A ction de la p a ra th o rm o n e dans 1^ m é tab o lism e pho sp h o - -ca lc ique . L a P re sse M édicale, 63: 1320— 1324.

99. R a u c h , S., 1959: D ie S p e ich e ld rü sen des M enschen. G. T h iem e — S tu t tg a r t .100. R a y n a u d , J., 1954a: N ouvelles o b serva tions c o n c e n tra n t les tr a n s fo rm a tio n s

ap rès c a s tra tio n , de la g lan d e so u s -m a x illa ire de la souris m âle . C. R. Soc. Biol., 148: 1743— 1747.

101. R a y n a u d , J., 1954b: E ffects de la su rrén a lec to m ie , associée à la c a s tra tio n su r la s tru c tu re de la g lan d e so u s -m a x illa ire de la sou ris m âle . C. R. Soc. Biol., 148: 1939— 1942.

102. R o b e r t i s de, E. D. P., 1940: T he cytology of th e p a ra th y ro id g lan d of ra ts in je c te d w ith p a ra th y ro id e x tra c t. A nat. Rec., 78: 473—495.

103. R o b e r t i s de, E. D. P., 1941: T he cytology of th e p a ra th y ro id and th y ro id g lan d s of ra ts w ith e x p e rim e n ta l rick e ts . A nat. Rec., 79: 417.

104. R o b e r t i s de, E. D. P., N o w i ń s k i , W. W. & S a e z, F. A., 1949: G en era l C ytology. 1—456. P h ilad e lp h ia .

105. R o m e i s . B.. 1948: M ik roskoü ische T echn ik . M ünchen . 1—530.106. R o s o f, J. A.: 1934: A n e x p e rim e n ta l s tu d y of th e h is to logy an d cy to logy of th e

p a ra th y ro id g lan d s in th e a lb ino ra t . J. exp . Zool., 68: 121— 165.107. R u c a r t, G., 1949: C lass ifica tion e t v a lle u r fo n c tio n n e lle des ce llu les p a ra th y r -

o ïd iennes des m am m ifè res . A rch . A nat. M icr. M orph. E xpér., 38: 1.108. S a n d s t r o m , J., 1880 (acc. to K rook , 1957): U p p sa la L â k F o ren . F o rh ., 15: 4.41.109. S a n n a z z a r i , P. , B e l l o n i , L. & M e n o z z i, P . G., 1959: H is to m o rp h o lo -

g ica l m od ifica tio n of p a ra th y ro id s fo llow ing a d m in is tra tio n of P T H . A rch . E. M arag lian o P a t. C lin., 15, 5: 1499— 1504.

110. S a s s a k i , T., N a k a j i m a, H., K a n d a, K. , A r i y o s c h i , T. & M o c h i - z u k i, H., 1953: T re a tm e n t of ch o n d rodystroph ia fo e ta lis and re f ra c to ry r ic k e ts w ith p a ro tin . A cta P a e d ia tr ic a Jap ., 57: 584.

111. S a w i c k i , W., 1960: O w ew n ę trzn y m w y d z ie lan iu ś lin ian ek . W iad. lek., 13: 797— 805.

112. S a w i c k i , W., 1961: T ra c e r s tu d ies on th e fu n c tio n a l re la tio n b e tw e e n sa liv a ry g lan d s and th e th y ro id . B ull. A cad. Pol. Sci., cl. II , 9: 337—339.

113. S c h n e i d e r , R. M. & P e r s o n , P., 1960: A erob ic o x id a tiv e m e tab o lism of s a l iv a ry g lands. A nn . N ew Y ork A cad. Sc., 85: 201—207,

254 I. D zie rżykray-R oga lska

114. S c h n e y e r , L. H. & S c h n e y e r , Ch. A., 1960: R eg u la tio n of sa liv a ry g land am ylase ac tiv ity . A nn. N ew Y ork A cad. Sc., 85: 189—200.

115. S h a f e i', W. G. & M u h 1 e r, J. C., 1960: E n docrine in fluences upon th e s a l iv a ry g lands. A nn. N ew Y ork A cad. Sc., 85: 215—227.

116. S h i b a t a, K ., N e g i s h i, A., S a i t o, K. & T a d o k o r o, S., 1954: E ffec t of sa liv a ry g lan d -ec to m y on th e response of th e tib ia l ep iphysis o t g ro w th horm one. E ndocr. Jap ., 1: 203—207.

117. S n e l l , G. D., 1956: B iology of la b o ra to ry m ouse. D over p u b lica tio n Inc.: 1— N ew Y ork .

118. S o s z k a , S. & K r a w c z u k , A., 1960a: O w p ływ ie n iek tó ry ch czynników na ob raz m orfo log iczny ją d ra szczura białego. U szkodzenie ś lin ian k i p rzyuszne j. G inek . poi., 31: 501—508.

119. S o s z k a , S. & K r a w c z u k , A., 1960b: W pływ uszkodzen ia ś lin ian k i p rz y usznej n a o b raz m orfo log iczny ja jn ik ó w szczu ra b iałego. P am . X IV Z jazd u G inekologów . P ań stw . Z akł. W yd. Lek., 775—786. K ryn ica .

120. S r e e b n y, L. M., 1960: S tud ies of sa liv a ry g land p ro teases. A nn. N ew Yoi'k A cad. Sc., 85: 182— 188.

121. T a k i z a w a , N., 1954: A p a tho log ica l re sea rch on th e in te rn a l secre tion of th e sa liv a ry g lan d s . A cta P a th . Jap ., 4: 129— 166.

122. T a 1 m a g e, R. V. & D o d d s , B. F., 1955: C o m p ara tiv e s tu d y of som e effec ts of a d m in is tra tio n of d ih y d ro tach y s te ro l and ca lc ife ro l to ra ts . E ndocr., 57: 236.

123. T r e m b l a y , G. & P e a r s e, A. G. E., 1959: A cy tochem ica l s tu d y of o x y d a tive enzym es in th e p a ra th y ro id o x yph il cell and th e ir fu n c tio n a l sign ificance . B rit. J. e x p tl. P a th ., 40: 66—69.

124. W a l k e r , R., 1958: Age changes in th e r a t ’s e x o rb ita l la c r im a l g land . A nat. Rec., 132: 49— 69.

125. W a l t h a r d , B., 1961: E lec tron -m icro scop ica l s tu d y of th e p a ra th y ro id s . A rch . De V ecchi A n a t. P a th . U niv. B ern . (Exc. Med. Sec. I, 15: 500).

126. W e l s c h , R., 1898: C oncern ing th e p a ra th y ro id g lands. J. A nat. Physiol.127. W e y m o u t h , R. J., 1957: T he cytology of th e p a ra th y ro id g lands of th e r a t

a f te r b ila te ra l nep h rec to m y , ad m in is tra tio n of p a ra th y ro id ho rm one and h y p o - physectom y. A nat. Rec., 127: 509—525.

128. W i ś n i e w s k i , L., 1960: O w pływ ie trz eb ien ia n a ob raz m orfologiczny p rz e d n iego p ła ta p rzy sad k i m ózgow ej, g ruczo łu tarczow ego , p rzy ta rczy c o raz n a d nerczy m yszek b ia łych . E ndokr. poi., 11: 299—312.

129. V a 1 e r i, V. M., 1954a: N uclear vo lum e and te s to s te ro n e -in d u ced changes in sec re to ry ac tiv ity in th e su b m ax illa ry g land of m ice. Science, 120: 984— 985.

130. V a l e r i , V. M., 1954b: A ction de la te s to s te ro n e su r le vo lum e n u c lé a ire d an s la g lan d e so u s -m a x illa ire s de la so u ris . Com p. R end. S. A cad. Sc., 238: 1613— 1615.

131. Y a n g , M. F., 1960: O n th e changes of h is to log ical s tru c tu re , especia lly glycogen co n ten ts of th e p a ra th y ro id g land of th e w h ite r a t d u rin g p regnancy an d p u e r - pe riu m . A c ta S cholae Med. in G ifu , 8.

132. Z a w i s t o w s k i , S t., 1962: C y tochem ickâ s tu d ie o n e k te rÿ c h enzym ech v p a ra - th y reo id e i k ry sy za ruznÿch pokusnÿch stavu . C eskoslov. M orfol., 10: 205— 212.

M edical School,D ept, of H isto logy ,B ia ły stok , K iliń sk iego 1.

Pow iązan ie czynności ś l in ianek p rzyusznych i p rzy ta rczycy 255

ST R E SZ C ZE N IE

A u to rk a za jm u je się zależnością m iędzy p rzy ta rczy cam i a ś lin ian k am i p rzy u szn y - m i — g ruczo łam i zw iązanym i z g o sp o d ark ą fo s fo ro w o -w ap n io w ą o rg an izm u . W tym celu p rz e b a d a ła :1. W pływ u su n ięc ia p rzy ta rczy c n a o b raz m orfo log iczny ślin ian ek p rzyusznych .2. W pływ u su n ięc ia ś lin ian ek p rzy u szn y ch n a obraz m orfo log iczny p rzy ta rczyc .3. W pływ p o d aw an ia p a ro ty n y n a o b raz m orfo log iczny p rzy ta rczy c i ś lin ian ek p rz y usznych.4. W pływ u su n ięc ia ś lin ian ek oraz p o d aw an ia p a ro ty n y n a kości szczurów . B ad an ia p rzep ro w ad zo n o p rzy użyciu P 32.5. D odatkow o p rzep row adzono an a lizę s ta ty s ty c z n ą opracow anego m a te r ia łu .

P o u su n ięc iu p rzy ta rczy c w ślin ian ce p rzy u szn e j szczu ra w y s tę p u je zan ik tk a n k i g ruczo łow ej n a rzecz tk a n k i tłu szczow ej, co m oże św iadczyć o w yco fy w an iu się p ew nych p a r t i i m iąższu gruczołow ego z czynności.

W w y n ik u u su n ięc ia ś lin ian ek o trzy m an o w p rzy ta rczy cach n a s tę p u ją c e zm iany: p o w iększen ie się w y m ia ró w k o m ó rek gruczo łow ych i ich ją d e r , obecność licznych podz ia łów kom órkow ych , s ta łe u łożen ie ją d e rk a w pob liżu b łony ją d ro w e j o raz zw ięk szan ie się ilości k o m ó rek ja sn y ch — czynnych . Pow yższe d an e p o zw a la ją n a w yciąg n ięc ie w n io sk u , że p rzy ta rczy ce po u su n ięc iu ś lin ian ek w y k azu ją cechy pobudzen ia .

P o p rzep ro w ad zen iu dośw iadczeń po lega jących n a p o d aw an iu p a ro ty n y uzyskano: zm n ie jszen ie s ię ją d e r kom órkow ych , zm n ie jszen ie się p iro n in o -ch ło n n o śc i p ro to - p lazm y, pow iększen ie się ilości tk a n k i łącznej w p rzy ta rczy cach o raz zm n ie jszen ie się k o m ó rek i ją d e r k o m órkow ych w p ęch erzy k ach ś lin iankow ych .

O b se rw ac je te św iadczą o osłab ione j czynności zarów no p rzy ta rczy c ja k i ś l in ia n ek u szczu rów tra k to w a n y c h p a ro ty n ą .

Do b ad ań au to rad io g ra fic zn y ch uży to szczury z w y cię ty m i ś lin ia n k a m i o raz szczury , k tó ry m po d aw an o p a ro ty n ę . P rzy użyciu rad io ak ty w n eg o P 32 b ad an o w p ły w po w yższych czy n n ik ó w n a poziom fo sfo ru w kościach szczurów . N a jw ięk szą ak tyw ność w pop ie le k o stn y m uzyskano u zw ie rzą t, k tó re o trzy m y w ały 0,2 m g p a ro ty n y n a 100 g w ag i c ia ła w ciągu tygodn ia . W ynikom ty m o dpow iada o b raz au to rad io g ra ficzn y . N a jm n ie jsz ą ilość im pu lsów stw ie rd zo n o w pop iele k o stn y m zw ierzą t, k tó re zostały sek c jo n o w an e w tydzień po u su n ięc iu ś lin ian ek , czem u o dpow iada ob raz a u to ra d io g raficzn y .

P rz e p ro w a d z o n a ana liza s ta ty s ty c z n a opracow anego m a te ria łu um o ż liw iła s tw ie r dzen ie , że: a) w p rzy ta rczy cach zw ie rzą t z u su n ię ty m i ś lin ia n k a m i w y s tę p u ją w ię k sze ją d ra kom ó rk o w e w p o ró w n an iu ze zw ie rzę tam i k o n tro ln y m i, b) pod w pływ em p a ro ty n y w p rzy ta rczy cach zw ie rzą t n ie sp o ty k a się ją d e r za rów no bard zo dużych ja k i b a rd zo m ałych . W ydaje się, że p a ro ty n a d z ia ła n a u jed n o licen ie p rzec ię tn y ch ro zm ia ró w ją d e r .

J a k w y n ik a z pow yższych danych , u sun ięc ie p rzy ta rczy c spow odow ało zm iany m orfo log iczne w ś lin ian k ach p rzy u szn y ch św iadczące o w y co fan iu się ty ch g ru czo łów z czynności.

Z d ru g ie j s tro n y u sun ięc ie ś lin ian ek , w yw ołało nadczynność p rzy ta rczy c , a p o d aw an ie p a ro ty n y spow odow ało zab lokow an ie czynności p rzy ta rczy c .

W y d a je się, że pow yższe w y n ik i w sk a z u ją n a is tn ien ie d z ia łan ia zw ro tnego cbu g ruczo łów z tym , że p rzy ta rczy czk i są g ruczo łem n ad rzęd n y m (p ro d u k to rem ) w s to su n k u do ś l in ia n k i p rzy u szn e j (efek to ra).

Z p rzep ro w ad zo n y ch dośw iadczeń w y n ik a rów nież , że ś lin ian k i w y w ie ra ją sw ój w p ły w n ie ty lk o n a p rzy ta rczy ce , ale ró w n ież n a tk a n k ę ko stn ą . U sun ięc ie ś lin ian ek

256 I. D z ie rżykray-R ogalska

przyusznych p o w o d u je bow iem ch a rak te ry s ty czn e obn iżen ie się poziom u P 32 w kości, podczas gdy p o d aw an ie p a ro ty n y w p ływ a n a w zrost zaw arto śc i P 3-\ W oparc iu o o trzy m an e w y n ik i a u to rk a w ysuw a dw ie h ipo tezy , do tyczące w p ły w u ś lin ia n e k na kość. W edług p ie rw sze j z nich ś lin ian k a p rzy u szn a w p ły w a bezpośredn io n a tk a n k ę ko stn ą , w ed ług d ru g ie j — poprzez gruczoły p rzy ta rczycow e, k tó re ja k w iadom o d o tychczas w p ły w a ją d ecydu jąco n a p rzem ian ę fo sfo row o-w apn iow ą.

EX PLA N A TIO N OF PL A T E S

P la te X.

Fig. 1. P a ro tid sa liv a ry g land. C ontrol. L arge am o u n t of RN A in th e basic p a r ts of th e v e s icu la r cells. M agnified 450 X.

Fig. 2. S tr ia te d duct. C ontro l. S tr ia tio n in th e basic p a rts of th e cells. M agn. 1160 X.Fig. 3. P a ro tid s a liv a ry g land (after rem o v a l of p a ra th y ro id gland). In te r lo b u la r

duct. T he in n e r la y e r of the ep ith e liu m flak es of in to th e lu m en of th e duct. M agn. 150 X.

Fig. 4. P a ro tid sa liv a ry g land (after rem oval of p a ra th y ro id g land). In ad d itio n to n o rm ally fo rm ed lobes, th e re are lobes of a lte re d s tru c tu re . M agn. 150 X.

P la te X I.

Fig. 5. P a ro tid g lan d (a f te r rem oval of th e p a ra th y ro id g land). T he vesicles have a la rg e lu m en s ligh tly sim ila r to the vescles iof th e th y ro id g land . L u m en of vesicles filled w ith secretion . M agn. 450 X.

Fig. 6. P a ro tid sa liv a ry g land (a fte r rem o v al of th e p a ra th y ro id g land). In th e a lte red lobe (righ t) th e cy top lasm of th e cells ex h ib its in ten siv e vacuo lisa tion . M agn. 300 X.

Fig. 7. P a ro tid s a liv a ry g land (a fte r rem oval of th e p a ra th y ro id g land ;. C onnective tissu e w ith a la rg e n u m b er of fa t ty cells p e n e tra te s b e tw een th e a lte re d vesicles. M agn. 150X.

Fig. 8. P a ro tid sa liv a ry g land (a fte r rem o v a l of th e p a ra th y ro id g land). C on tinued p rocess of p e n e tra tio n by th e connective tissu e w ith s im u ltan eo u s a tro p h y of th e g la n d u la r vesicles. M agn. 150 X.

Fig. 9. P a ro tid s a liv a ry g lan d (a fte r rem oval of th e p a ra th y ro id g land). L obes of connec tive tis su e have been fo rm ed in p lace of th e g lan d u la r lobes, in th em are th e re m a in in g s tr ia te d ducts. M agn. 150X.

P la te X II.

Fig. 10. P a ra th y ro id g lands of a con tro l ra t . T he nucle i e x h ib it co n sid erab le p o ly m orph ism . M agn. 1160X.

Fig. 11. P a ra th y ro id g land a f te r rem oval of th e sa liv a ry g lands. L ig h t cells a rra n g e d in p seudo -vesic les w ith a sm all lu m en in th e cen tre . C y top lasm filled w ith fine g ra n u la r m a tte r . F ixed by L ison -V oaker. M agn. 1160X.

Fig. 12. P a ra th y ro id g lan d (a fte r rem oval of th e sa liv a ry g lands). M itoses. M agn. 1160X.

Fig. 13. P a ra th y ro id g land (a fte r rem oval of th e sa liv a ry g lands). PA S + g ra n u la r m a tte r in th e cells ly ing in the p e rip h e ry . M agn. 1160X.

P aro t id sa l iva ry g lands and p a ra th y ro id g lands 257

P la te X III .

F ig . 14. P a ra th y ro id g land (a fte r rem o v a l of th e s a liv a ry g lands). L a rg e G olgi a p p a r a tu s, fo rm ed of th ick trab ecu lae , ly ing in th e ap ica l p a r t of th e cell. M agn. 2200 X.

F ig . 15. P a ra th y ro id g lan d (a fte r rem o v a l of th e sa liv a ry g lands). H igh cy lin d rica l cells, s i tu a te d w ith th e ir base on b lood vessels. M agn. 1160X.

F ig . 16. P a ra th y ro id g lan d (a f te r rem o v a l of th e sa liv a ry g lands). C ells hav e ac id i-ph ilous cy top lasm and h y p e rch ro m a tic nu le i. N uclei becom ing n a rro w e r(bottom left). M agn. 1160 X.

F ig . 17. P a ra th y ro id g lan d (a f te r rem o v a l of th e sa liv a ry g lands). T he G olgia p p a ra tu s , s i tu a te d on th e ap ica l pole of th e cell, is fo rm ed of de lica te sing letra b e c u la e and sm a ll frag m en ts . M agn. 2200X.

P la te X IV .

F ig . 18. P a ra th y ro id g lan d (a fte r p a ro tin in jec tions). O ne k in d of cell w ith d a rk cy top lasm and v a rifo rm nuclei. D is tin c t red u c tio n in d im en sio n s of nuclei. (P e r ip h e ra l p a r t of g land). M agn. 1160X.

F ig . 19. P a ra th y ro id g lan d (a f te r p a ro tin in jec tions). T he G olgi a p p a ra u s ly ing in th e ap ica l p a rts of th e cells is sm a ll and sca tte red . M agn. 2200 X.

P la te XV.

F ig . 20. P a ro tid sa liv a ry g land (a fte r p a ro tin in jec tions). E n la rg ed b lood vessels and ly m p h a tic vessels n e a r th e s tr ia te d d u c ts . M agn. 450 X.

F ig . 21. P a ro tid sa liv a ry g lan d (a f te r p a ro tin in jec tions). E n la rg ed blood vessels n e a r th e s tr ia te d ducts . M agn. 450 X.

F ig . 22. P a ro tid s a liv a ry g lan d (a f te r p a ro tin in jec tions). R ed u c tio n of R N A in th e basic p a r ts of th e vesicles. M agn. 450 X.

F ig . 23. P a ro tid sa liv a ry g land (a f te r p a ro tin in jec tions). P A S 4- g ra n u le s v is ib le in th e in te rc a la r ducts . M agn. 450X.

P la te X V I.

F ig. 25. A u to rad io g ram of th e base of a bone in a co n tro l ra t .F ig . 26. A u to rad io g ram of th e base of a bone from a r a t k illed 7 days a f te r excision

of th e sa liv a ry g lands.F ig . 27. A u to rad io g ram of th e base of a bone from a r a t k illed 14 days a f te r excision

of th e sa liv a ry g lands.F ig . 28. A u to rad io g ram of th e base of a bone from a r a t k illed 21 d ay s a f te r excision

of th e sa liv a ry g lands.Fig. 29. A u to rad io g ram of th e base of a bone from a r a t w h ich h ad been g iven

0.1 m g. p a ro tin .F ig . 30. A u to rad io g ram of th e base of a bone fro m a r a t w h ich h ad been g iven

0.2 mg. p a ro tin .

P A Ń S T W O W E W Y D A W N I C T W O N A U K O W E * W A R S Z A W A 1963

N ak ład 1450 egz. Ark. w yd . 4,25. M aszyn op is o trzym an o 19.V II .1963. P o d p isa n o do druk u 2.XII.1963. D ru k u k oń czon o 10.X II. 1963. P ap ier

druk sat. kl. III 80 g. F orm at B5. Cena 15 zł

b ia ło s to c k ie Z ak łady G raficzn e , z a m . 2780. * A-2.

A C T A T H E R IO L O G IC A , Vol. VII, 12. P la te X.

I. D z ie rzykray-R oga lska auctor phot.

ACTA T H E R IO LO G IC A , Vol. VII, 12. P la te XI.

I. D z ie rzy k ray -R og a lska a u c to r phot.

A C T A T H E R IO L O G IC A , Vol. VII, 12. P la te X II.

I . D z ie rz y k ray -R o g a lsk a aucto r p h o t.

A C T A T H E R IO LO G IC A , Vol. VII, 12. P la te X II I .

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-i « P a

I. D z ie rzyk ray -R o ga lsk a anctor pho t.

A C T A T H E R IO L O G IC A , Vol. VII, 12. P la te X IV .

I. D z ie rzy k ray -R og a lska uuctor pho t.

ACTA T H E R IO LO G IC A , Vol. VII, 12. P la te XV-

M r . , Л trf. ' M S t ^ \ >. 4 , ; * '» • / v ; —¡ r ■ V v . *

■ ’ í¿ *$5* T i e e t i r 4 ** ^. ^ r -> á k > ^ ? .;. f l H i . А Г - *: *y‘-1* З ч > ♦

r A a V ä v ^ - * ^^ j f ' ^ ‘ . . :v £ : л

<4 •' * -• О /* * - . .* '

I . D z ie rzy k ray -R o ga lsk a auctor ph'Dt.

A C T A T H E R IO L O G IC A , Vol. VII, 12. P la te XVI.

I. D z ie rz y k ra y -R o g a lsk a aucto r phot.

BIBLIOTEKA Instytutu Biologii Ssaków Polskiej Akademii Nauk

N rC z. 40 .2

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