INTKhNUCLEAB CRYSTALS I N THE IIKPATIC CELLS OF (IANIDSE-~rOT,VF',X, FOXES, JACKALS

AND NON-I)031F;STTCi DOGS

IIAROLD L. TEATHERFORD 7 ) f ~ p ~ z r t m r nt of Anatomy , HawaiYl Xedicul School, Boston, Xassnc1airsrtl.F

ONE PLATE (T?.VEL'I'E FIGURES)

117 a previous study (Weatherf ord, '38) on intranuclear crystals in the hepatic cells of the dog, variations were noted in the number of nuclei with crpstals-0 to 2.137G-aii average of 1.039). Except in the proximal convolntd tuloules of the kidney--a finding shared by lilar.cliand ( 1887 and '09), Grandis (1889), Rraiidts ( '09>, Berg ( '29) , aiid Nicolau and Iiopcio~lr- ska ( '36)-intrannclear crystals were not observed in other tissues of the dog. Grandis (1889) was unable to fiiid intra- nuclear crystals in the frog, turtlc, pigeon, gomg rat, rabbit (normal and dead from inanition), sheep, cow, young cat, pig and man.

The only meiitioii of intranuclear crystals in hppatic cells other than in the dog, so far as 1 am awarc, mas by Berg ('34) in a fox (species not given), an observation suggesting that these crystals ma? be found in still other Canidae.

I am grcatlp indebted to Dr. I'ierbert Fox, pathologist t o the Zoological Society of Philadelphia, who sent me the blocks of liver of repi'esentative Canidae, which made this study possible. The Cariidae examined were : prairie wolf (Canis latrans) aiid gray fox (Urc.\-on ciiiereo-al.gentens) two speci- mens, American red fox (Vulpes fulva), Fennw fox (F'eiiiiecus zerda), North African jackal (Thos anthus), Cape hunting dog (Lycaon pictus), a d the h s h dog (Icticyon venaticus)

29

THE Ih'4T03IlCAII RECORD. T O 1 73 , NO. 1

30 H. L. WEATHEKFORD

one specinieii each1 The material, already embedded in paraffin, was sectioned 5 p thick and mounted serially. Sec- tions on alternate slides were stained with hematoxJ-lin and eosin, and with Heidenhain’s ‘azan.’

OBSERVAT ross Forty-five thousand nuclei are counted--5000 in each liver,

1000 in a section-from rach of the Canidac examined. The findings are presented in table 1.

As in domestic dogs, the number of nuclei containing crystals is variable. Even members of the same species

mnLx 1

representative Canidae tubukztton of l l i e number of nurrc1 rontnznzlig crystak i n the !tiers of

SPECIXEIC

Prairie wolf Prairie wolf Gray fox Gray fox dmerieaii red fox Fennee fox North African jackal Cape hunting dog Bush dog

NUCLEI COUNThD

5000 5000 5000 .5000 5000 5000 5000 5000 5000

NUOLEI WITH CTWSTALS

7 15 36 13 4

49 10

5 10

PER. CEWT CXSSTALS

0.14 0.30 0.52 0.26

0.98 0.20 0.10 0.20

0.08

show considerablc differences in crystal content. It is noted that in the two specimens of prairie wolf one contains more than twice the number of nuclei with crystals than the other : the percentages, however, are lorn in both wolves. The same is true of the two specimens of gray fox, although in each of these the percentages of nuclei enclosing crystals are higher than in the prairie wolves. In the livers of two non- domcstic dogs--the Cape hunting dog and the bush dog- the crystal content is extremely low; in fact the percentages of nuclei with crystals are coiisiclerably lower than in the average domestic dog. There are even fewer than reported in a previous study on fonr pure bred Dalmatian dogs-

% Tlie terminology follows suggestions froin Dr. Glov-er 31. Allen, as perlisps that most generally acreptrd today.

INTRANUCLEAR C R P S T d L S IN H E P A T I C CELLS 31

tlie breed consistently showing lorn counts of nuclei wit11 crystals. The single specimen of Fennec fox shot\-s the highest count-O.98~-most nearly approaching the average found in the domestic dog.

Crystallographically the intranuclear crystals are similar in all Canidae. They are prismatic, with hexagonal cross sections, and occup;~ clear areas or ~-acuoles wi thiii niiclei. Despite similarities in crystal form, species differences are observed. In some species the crystals are short and broad, while in others they are long and narrow. The longest crystals found, however, are usually shorter than many of those seen in livers of domestic dogs. Seldom do the:- have lengths equal to the diameters of tlie nuclei enclosing them. In the foxes the crystals are narrow and often occur miiltiple in tho same or jn separate clear areas, ~-1iile in the jackal they are wider, generally single, sometimes elongating the nucleus. Only small crystals are found in tlie Cape hunting dog. The largest single cq-stal observed in any of tliese Canidae is in a specimeii of Anierican red fox whcre it dis- tends the envelope surrounding the vacuole enclosing it into an elongaled ellipse nearly equal to the diameter of the cell

Crystals arc observed in nnclei of several sorts-those of average size and goo4 staining, l a r p and pale 011~3, and small and dark staining ones. Differences in physiologic and mild pathologic conditions do not appear to affect the crystals. They are seen in cells ~ - i t h clondy swelling, pig- mentation, and in areas of focal necrosis. No crystals are found, however, in regions shoving more extensive pathology.

Besides the intrannclenr crystals some nuclei disp1a:- homo- geneons, rounded, ovoid and irregular. bodies. These bodier, while usually occupying separate clear areas, may be found in the same areas with crystals. The homogeneous bodies are prcsent with g e a t e r frequency in thc nuclei of hepatic cells in some Canidae than in others. They occur abundantly in the prairie wolf and Cape liunting dog but are not quite so numerous in the red fox and North African jackal. I n the Cape hunting dog, the homogeneous bodies are observed

(fig. 4).

32 H. L. WE.kTHERFORD

as oftcii as the iiitranuclear crystals, and in some c~ounts of 1000 nuclei mow often. Sicyificaiitly, homogeneous bodies are met with but rarely in both nuclei of hinucleate cells, and a similar condition prevails fo r the intranuclear crptals . Some nuclei exhihit an apparently empty clear area or vacuole. Whether this is a real coiidition or one resulting from cutting is not determined.

The sizes of' tlie homogcweous in twaiiuclear bodies vary j some are small, no larger than a true iiucleolus, while others are olnserved having one-third to one-half tlie diameter of the nuclens. Like the crystals tlic homogciieous hodies do not fill completely the clear areas enclosing them.

Illtralluclear crystals and homogeneous bodies stain simi- larlj-. Both stain with acidic. (eosiii) and basic (azo-carmine) dyes. As in livers of domestic dogs, which mere stained with a rarietr of dyes, there is observed neither pronounced a ciilopliilia no r bas ophilia.

The development of intranuclear cr>-stals is studied best in the foxes and tlie (-ape hunting dog. In the livers of these Canidae marked yariations in crh stal size are displayed, rang- ing from dispersed graiinlations near tlie limits of distinct microscopic resolution to wTell-formrcl crystals as long or long.ei* than the diameter of a nucleus. Xegardlcss of size the hexagonal prismatic shape prevails in all crystals.

DTSCUSRIOS

Observations show the presence of intrtmuclear crystals and homogeneous hoclies in the hepatic cells of nine members, representing sewn different species of the f amil j - Canidae.

Berg ( '34) discussed possible relationsliips between homo- geneous intranuclear bodies and cryvstal formation. IIe as- sumed that material for the crystals wandered in thr.ougli the iiuclcar membrane, concentrated in vacuoles as fluid drop- lets, and formed liomogeiieous bodies from which the cq-s tals a,i*ose. Vacrioles were stated to be present in hepatic cell nuclei of forms other than the dog, both cold blooded animals aiid mammals, and it was conjectnrecl that a lack of concen- tration or thc presence of some disturbing substances liinclcrd

INTIIANUCLEhIi CRYSTALS I N HEPATLC ChLLS 33

cryst;illizatioii in tliese cuscs, though the gcneral metabolic conditions might be similar. Deficient concentration in the fluid droplets of the hepatic cell nuclcus may account for the conditioii, \~r)- iiotic*eaWc in the C u p liinrtiiig dog, \ vhc~uc~ tlie homogeneous bodies are especiallp ~iumcrous and llie intiminclclar crystals few. It is impossible, however, to state whether tliesc homogciieoiis bodies have any relation to the inlranuclcar iiiclnsioii bodies, presumably produced by fillra- blc vin.mes, clescribed hy Cowdry aiid Scott ('30) and Nicolaii and Kopciomska ( ' 3 6 ) in the hepatic cells of dogs, though the latter authors on not too secure a basis, similarity in staining, sought to derive the iiitranuclear cr;vstals f rom the inclusioir bo rli e s .

From studics of illustratioiis, in a iiumber of papers, cle- pictiiig iiitranuclear inclusion bodies, I beliw--e that while in some cases the figures s l i o ~ ~ ~ similarities to the homogeneous bodies seen iii hcpatic cell iiuclei of Canidae, iii others the>- present di8erenccs. Ludford ( '30) stated: "The intra- nncleau T-ii-iis bodies . . . . are remarkably alike. Xltlrough some occasionally stain faintly basophil, the majority stain readily with acid dyes.' ' Intraiiaclear crystals and homo- geiieoas bodies stain with either acidic o r hasic dyes. Even with hematosylin and eosin, when sections are stained deeply with hcmatoxylin the crystals take the basic dye and when stained less deeply the acidic dye. All of which leads me to thiiik that the staining of both of these formations is pos- sibly one of physical adsorption.

Cowdry ('28) lias given some details of microchemical studies on intrannclear inclusions and wliilc showing results, in part, comparable to those of Berg ('29) and mine ('38) on intranuclear crystals, they do not prove concln~ively that these two arc of the same material. More work should be done on 1mth l o extricatr tlic proldem, and the suggestions of Ludford ('30) might be followed profitably. Tlic m e of the ultra- centrifuge, as by LLVXS ( '36) investigating intraiiuclear in- clusion bodies, sliould he tried also in the study of iiitra- nuclear crystals.

Some of the earlier investigators, largely bccause of simi- larities in staining, considerccl the intranuclear crystals as

34 €1. L. WESTHERFORD

hemoglobin or a derivative. Crystallograpliicall~~ the intra- nuclear crystals in livers of domestic dogs and in other mem- bers of the family Caiiiclae a re dissimilar to hemoglobin.

Reiclicrt and Brow11 ( ’09) studied the crystallography of hemoglobin in 1 m l v e members of the family, ten distinct species and two varieties or crosses. A general type of crystal was observed common to all species. It was described as more or less an eloiigated prism with a diarnond-shaped cross section a i d nsually strongly striated longitndinally. TTThile t i e ratio of length to thickness of the crystals varied, both in the same and in different species, the crystallographic characters were fourid to bc similar.

Because of the diffewcnces in form of hemoglobin aiicl iiitra- nuclear crystals in all tlie obserrcd (’auidae, and the absence of iron in the ash of the intrannclear crystals in clogs- pointed out by Qrandis (lass), Berg ( ’29) , and Weathcrford (38)-it is ~ i i i l i k~ ly that iron vil l be found in similar intra- nuclear c r y t a l s in other members of the family. Therefore it is concluded that the intrannclear crystals are not Iicmo- gl obin.

A . Hunter and his co-worlicus (’1.1. and ’19), in their com- parative studies of purine metabolism in various represeiita- tive mammals, reported ‘uricolybic indexes’ of 98, 97, 96 and 32, respectively, for tlie domestic dog, prairie wolf (coyote), dingo, and Dalmatian dog. This indpx is the ratio of the output of allaritoiii nitrogen to thc sani of the output of al- lantoin and uric acid nitrogen.

From counts of hepatic cell nuclei in the present study, and in an earlier one on dogs, i t is seen that those ipccies ir-it11 the highest percentage of nuclei containing crystals have the highest ‘uricolytic index,’ atid those with tlie fewest crystals the lowcst index. These findings suggest a probable relationship between purine metabolism and crystal formation.

Perhaps long domestication of dogs, with attendant meta- holio changes, acconiits in part for the greater power of crystallization and tliercfore the more numerous and usually larger intramclear c+rl\-stals than arc observed in other Clanidae.

INTJL2NUCLEXLt CL~YSTATJS IIT HEPATIC CELLS 35

SUMMARY

1. Intranuclear crystals are observed in the hepatic cells of nine individuals, representing seven species of Canidae. The number of nuclei with crystals varies in the different species from O.OSy& in the American red fox to 0.98% in the Feiinec fox.

2. In all members of the family the crpstals arc presented as hcsagonal prisms within clear areas or vacuoles. Crystal sizes range from the minute to those long enough t o distort the nucleus.

3. In some nuclei, liomogciieous, rounded, ovoid and ir- regnlar bodies are foulid, inclosed in separate or the same vacwole with erytals . Thew bodies am observed more often in some Canidae than ill others.

4. A comparison of the crystals 1%-ith what is known of the crystallography of hemoglobin in Canidae indicates that the two are not the same.

5. The observations on the intranuclear crystals, sugges- tions from thc ‘uricolytic index, ’ and previous csperimcdol studies lead to the conclusio~i that there is a relation betweeii purine metabolism and c r p t a l formation.

LITERATURE CITED

BERG., W. 1929 Zum mikroskopischeii Kachweis des Stoffmeclisels im Gewcbe. Die Kristalle in dcii Kcrnen der Lcber- und Nierenzellen des Hundes. Xtschr. f. mikr.-anat. Forseh., Bd. 16, S. 2113-238.

- 1934 CJber den mikroskopisch 1x4 rhweisharrii Ubcxrtritt von Stoffen aus dem Qtoplasma in cl,en Kern der Leberzelle. Ztschr. f . mikr.. aiiat. Forseh., Bd. 3.5, S. 146-380.

nRA4SDTS, C. E. 1909 c h w Einschliicsc iiii Kern Lebei-zelIiI iintl iliw Beziehungcn zur Pigmcntbildung a ) beini Hund, b) beim Meuschen. Beitr. z. path. Anat. u. z. nllg. Path., Bd. 45, S. 4 5 7 4 7 5 .

The microchenktry of nuclrar inclusions in virus diseases. Science, vol. 68, pp. 40-41.

A cornparkon of rel-tain iiitrsnue1ear inclusions found in the livers of dogs without history of infection Tit11 intranuclear inclusions characteristic of the action of filtrable viruses. Arch. Path., vol. 9, pp. 1184-1196.

GRANDIS, V. 1889 Sur certaines cristaiix qne l’on tronve dans Ic noyau des eellules du rein et du fnie.

COWDRY, E. V. 1928

COWDRY, E. V., AA7) (3. n. SCOTT 1930

Arch. itnl. rle biol., T. 12, pp. 137-151.

36 H. L. WEATHERFORD

HUNTER, A,, hf. H. GIVFYS AND C. If. GuIoIN 1914 Studies in the biochemistry of purine metabolism. I. The excretion of purine catabolites 111 the uriiio of nlarsupials, rodents and Carnivora. J. Riol. Chem., 101. 18, pp. 387-401.

Purine metabolisiii in various repiesenta- tive mammals.

TJltraeentrifugntion of iiitranuclear inclusions in the sub- maxillary glands of gninea pigs a n d ground moles. Am. J. Path.,

LUDFORD, R. J. 1930 Cell inclusions. A sp te in of hacteriology in relation to medicine.

IfARCHAXD, F. 1887 Ueber die giftige Wirkung der ~ i lo r sau ren . h c h . f . exp. Path. 11. Pharmakol., Bd. 23, A. 273-315.

1909 Hemerkuiig uber dip ' Keriikristalle. ' Beitr. z. path. Anat. u. z. allg. Path., Bd. 45. R. 507-509.

bur 1es manifestations d 'un ult ixtirus saprophyte clans l'organisme du cliieii. Ann. Inst. Pastcur, T. 57,

1%EICTiERT, E. T., AICD A. P. BROWTN 1909 The differentiation and specificitj of corresponding proteins and other TTital substances in relatioil to biological classification and orgaiiic er olutioii : the c r j s ta l lograpl i~ of ?i emoglobi 11s. Cariiegie Inst . Wa sliiiigt on, p~ibl. m o. 116.

WFATIIERFCIRD, H. L. 1938 -4 morphological and expcrinirntnl Rtudy of the iiitraiiuclear crystals i n the hepatic cells of the dog. Aiiat. Kec., aol. 71, pp. 413-445.

HTJNTER, A., AND F. V. WARD

LUCAS, A . M. 193G

1919 Tr. Roy. Roc. Canada, Sect. IV, vol. 13, pp. 7-11.

vol. 12, pp. 933-947.

Vol. 7, chap. 2, Med. Res. Coun., London.

NICOLATT, R., AND L. KOWIOWSKA 193(i

pp. 244-253.

P L A T E 1

EXPLAhTATION O F PIGLE3RS

Fjgiires 1 t o I?. Drawings of nuclei and contained crystals showing prismatic faces arid 11euagonxl cross ~ t ~ t i o n s . Observe the d e a r area surrounding the crystals.

1 to 3 4 to 5 (5 to 7 8 to 9

Prairie wolf (Canis la t ram) . American red fox (Vulpes fulba). Fennec f o u (Feiineeua zerda). Gray fox (TTrcvoii cinerco-argeiitrus).

North Sfricaii jacltal (Tlios anthus).

Cape hunting dog (Lyraoi i pictiis).

10 11 Rush dog (Icticyon wnaticus). 1 2

All figures are draivii in oiitline wit11 n camera lucida, details free-liand. The lens combination is n Zeiss apochroinatic oil im. ohj., 1.5 iu~ii., N.A. 1.3 and X 30 eomp. oc., X 3700.

INTRANtJCLEAR CIZYSTA1,S I N I IEPATIC CEI,T>S 11. 11. W C A T H R R F O R D

PLATE 1

37