Cell Biology International Reports, Vol. 4, No. 3, March 1980 265

THC CHRCG%T'3I.D EO9Y CF F.EIGSE SF'ERNA"i3S.

D.B. Krimer and P. Esponda

Instituto de Biologfa Celular (C.S.I.C.), Veldzquez,l44

Madrid-G, Spain.

ABSTFGCT

We have carried out a cyto'chenicai analysis of the chromatoid body employing preferential methods for the detection cf basic protezns and polysaccharides. The ChrOIil~tOiCi iiUi?y appcers ZDSitiVe after alcoholic PTA staining suggestiilg a basic protein compositicn.Vesic~es surrounding the chromatoid body appear positive after aqueous PTA and the Tlhiery procedure. The presence of polysaccharLdes inthe vesicles of the chrcmatoid body, and a rz1ationshi.p be-tween them and the Colgi complex is suggested.

T6.e Presence: 01' i:hll <hromatoid body has been reported dgr-ng the sgerp.atogcnesis zf severai species (Sud, .XGIa). It has lieen describei ii1 mammais - :a IJ"' -+ Y dense OrganCile !-;hiC!i is first observed during th; pachytene of themaiotic prophase, becoming most prom::?ent during the Gol.gi and eariy cap p&ses of spermiogenesis (Sud, i46la; FawcFb-t- et a-., 1 1970). The origin and fun.ztion Of the chrom,: t,sid body are not clearly understood and it has b:c~n suggeste,d t:T:at it could be derived fr0!T1

nuciiear X3 teriai jCcminc;s and Okadz., l.97?.) or dense interstitial material thataccumuiates in the mitochcndria of s~ermatocytes (Fawcett et al., 1970).

On the other hand, the observation of movements of the chromatoid body in the early spermatids of rats (Sbderstrtim and Parvinen, 1976s; Parvinenand Parvinen, 1979) seem to point to a possible exchange of material between the nucleus, the chromatoid body and the Golgi complex. This observationledthe authors to suggest that the chromatoid bodycculd play aroleinthetrans;mrtation of haploid gene products, including nucleocytoplasmic RNA, in the early spermatids.

03~1651/80/030265/~2. W/O @ 1980 Academic Press Inc. (London) Ltd.

Cell Biology International Reports, Vol. 4, No. 3, March 1980

Although several attempts have been madetodescribe the chemical composition of the chromatoid body there is still some disagreement regarding its make-up. The presence of RNAandbasic proteins havebeen demonstrated by means of some cytochemical techniques underthelight microscope (Sud, 1961b). However, various cytochemical procedures for the electron microscope have failed to indicatethepresenceofribonucleoproteins (Edciy, 1970). Nevertheless, SbderstrBm and Parvinen (1976b) after labelling with tritiated uridine and using electron microscope autoradiography,observed thatthechromatoid body appears strongly labelled only after along period of chase following the pulse. These results led the authors to suggest that the chromatoid body contains RNA which is synthesized in the nuclei of spermatids.

In this work we wish.to addnewinformation concern- ing the cytochemical properties of the chromatoid body by using some preferential techniques forthe detection of basic proteins and polysaccharides.

MATERIAL AND METHODS

The testes of adult mice were cut into small pieces and fi.xed in 3% glutaraldehyde in phosphate buffer pt! 7.2, for 2 hours at room temperature andthen postfixed in 1% osmil;n tetroxidein the same buffer. Some specimens were only fixedinglutaraldehyde.Thepi.eces were dehydrated throughanincreasing series of alcohols,andsoms of those fixed onlyinglutaraldehyde were stainedwith 2% alco- holic PTA,using thesheridan and Barrnett method.(19'39). All the samples WereembeddedinEpon 812. Ultrathin sections were stained with uranyl acetate and lead citrate. Samples with only aglutar- aldehyde fixation were stained with 2% aqueous PTA for20 minutes at rcom temperature (Stockert et al., 1975) or with silver pro- teinate after oxidation with thiosemicarbazide, following the Thiery procedure for polysaccharides (1967).

IiESULTS

The chromatcid body is more easily recognized in the YOUIl~ spermatids at the Golgi phase (Fig. 1). At this stage it appezirs close to the nuclear membrane and freqilently located near tne acrosomal complex. A large number of vesicles are frequently- observed associated with the chromatoid body (Fig. 2). These vesicles are preferenti.al located at the periphery although it is also possible to find them interming.led with the fibmllar material.

Cell Biology International Reports, Vol. 4, No. 3, March 1980 267

The chromatoid body has agreat affinity foralcoholic PTA (Figs. 3 and 4j. Fig. 3 shows that the highdegree of contrast of this structure appears similar to that observed in the condensed chromatin. Using aqueous PTA, apreferentialstaining of both the vesicles of the chromatoid body and those of the Golgi complex is achieved (Fig. 5and 7). The acrosomic granule, derived fromthc fusion of these vesicles,also shows considersble contrast as well a. s the material encircled in the acrosornic vesicle. In later stages, the acrosome is heavily stained (Fig. 6). These structures are stained in the same way using the Thiery procedure (Fig. 8).

The de-reloTlent Y L a' of new cytochemical techniques for

electron microscopy hasmadea better understanding Of

cell coinponents possible. The cytochemistry of the chromatoid body has been studied mainly using light microscopic techniques (Daoust and Clermont, 1955; Sud, 1961b). Rowever, a few cytochemical methods also have been applied at the electron microscope level (Eddy, i970). The alcoholic PTA, used in the present study, seems tobe apreferential staining for arginins-lysine rich histones (Sheridan and Barrnett, 1969). If this is the case, fRe positive staining of the chromatoid body would confirm the results obtained with the light microscope, suggesting the presence of basic proteins (Slid, 19613).

Concerning the vesicles surrounding the chromatcid body, there is not enough information a\Failable about its cytochemical properties. The positive reaction showed by- these vesicles employing botil the Thiery procedure andthcaqueau; p T .i m. s.2 t iTA $3 IjJ . , reveal 5 their 1;"' 'I- -Y sac ~~~~,rid-~ composition. Recently,a relations~?ipbet~~~?n the' Golgi complex and the chromatoid body has besn observed using electron microscope technique and cine- matography (Sdderstrbm and Parvinen, 1976; Parvinenand Parvinen, 1979). Our observation that the vesicles surroundinq the chromatoid body show a similar reaction to that observed at the vesicles of the Golgi ccmplex would also support such a relationship. The reason for this association is not clearly understood. However, it has been suggested that the chromatoid body would play some role in the formation of the acrosornal complex (Reger et al., 1977).

268 Ceil Biology International Reports, Vol. 4, No. 3, March 1980

Cell Biology International Reports, Vol. 4, No. 3, March 1980 269

8

Fig. 1. m-e.*- Early spcrmaiid in which t'ne chromatoid body is clearly ob- served (arrow) . "In viva" proparaticn observed with Nomarski. 1: = nucleus. s 2.500. Fig. 2. Electron micrographofthe chromatoid body of anearly spermatid. A !.arc;e num!zer of vesicles (arrox) can be seen surroundins the fibrillar portion. IJranyl acetate and lead citrate. :: 39.900. Fiq. 5.Early spermatid stained with alcohoiic PTA. <-..- TF-L chrome coid body appears intcnscly stained on cne side of the nucleus. ?I 2 nu- cleus; ch = chromarin. x 10.500. Fi?. 4. HigI: mzcrnification cf the chromatoid FGZFPTA. TheVvesicies are

body stained ~~Yjch a;co- not rrisualized with this staining pro-

cct!urc. x 29.000. Fia. 5. Acrosonic vesicles (vj ataj.ned vith atrucous PTA. A-- The Gcioi co;,~~~les is 0bser::e.d closely a.sSociat?d with <he 3cros3I31~ ;v>s;.cJe,> membrane. The sr.;zll vesicles cf t3e Gslgi co;~~!ex (arrows) 2 y-;-j :::qc proacro3:mic <ra::;lc f.cj arc clear:-J sraixd. x 43 .t?OO. Fiq. G.Lte qy-.r:3.cici stainad Vlzh aqusous PTA. T‘ne =-~cssx is strcn- L.-* --. sly CeI‘rl-~.~~eci. !, - r.l~CiEUS. x il. 5'30. Fig. 7 . ~7 11; 'L 0 7,; :z 3 i C PT';. Tile

> (; J xj (j f 1 ,) -2 ,J L 1 Ti, __:- ;>er.-natiJ. s';ai;;e5 w i tn &qi'"!,us

fi'~-i;ia~ r;artiOil cc the ChrJxatOid bGcy Sb;S;;S 2 ;',Zyy density hrtiil,a the &,a11 vesicles

1ci: (arrow-s) are stronTl..i s+;.i::-;:i.s 17 q-1~3. _I! ._C

Fit. D.Chrom~toi.3 hod; staiilcd xit.i the Tlliery pr:cci:-rc. T!?? smail vssicles (arrc;~:s; ar'-2 positively stair!-8j. :< !6.:?3.

270 Cell .Biology International Reports, Vol. 4, No. 3, March 1980

REFERENCES

Comings, D.E. and Okada, T.A. (1972) The chromatoidbody in mouse spermatogenesis: Evidence that it may be formed by the extrusion of nucleolar components. Journal of Ultrastructure Research 39, 15-23.

Daoust, R. and Clermont, Y. (1955)DistriEtion of nucleic acidingerm cells durinythecycleofthe seminiferous epithelium in the rat, American Journal of Anatomy 96, 255-283.

Eddy, E.M. (1970) Cytochemical observations on the chromatoid body of the male germ cells. Biology of Reproduction&, 114-128.

Fawcett, D.W., Eddy, E.M. and Phillips, D.M. (1970) Observations on the fine structure and relationships of the chromatoid body in mammalian spermatogenesis. Biology of Reproduction 2, 129-153.

Parvinen, II. and Parvinen, Z. (1979) Active movements of the chromatoid body. A possible transport mechanism for haploid gene products. J. Cell Biol. 30, 621-628.

Reger, J.F., Fain-Maurel, M.A. and Cassier P. (1977) The origin-,distribution and fate of the‘chromatoid body (germ plasm) during spermatogenesis and,spermio- genesis in two peracaridae. Journal of Ultrastructure Research 60, 84-94.

Sheridan, !J.E and Barrnett, R.J. (1369) Cytochemical studies on chromosc~m~ ultrastructure. Journal Of Ultrastructure Research 27, 216-229.

SddeCS trbz, K. and Parvineny-:I. (1976a). Transport of material Setwesn the nucleus, the chromatoid body and the Golyi conplexinthe early spermatids of the rat. Cell and Tissue Research 168, 335-342.

Soderstrbm, K. and Parvinerl, $1. (mbbj Incorporation of 3II) uridirie by the chrometoid i;ody during the rat spermatcgenesis. Journalof Cell Biology 70, 239- 246.

--

Stocker-t, J.C., Colman, O.D., Gim&'~c,z-I-iartfn, G. and Esponda, P. (3.9753 Selective staining of rodent acrosomes with phoaphotungstic acid. Mikroskopie 31, - 36-41.

Sud, B.N. (1961a) The chromatoid body in spermatogenesis. Quarterly Journal of Kicrcscopical Science 102, 273- 292.

Sud, B.N. (196lbj Morphologicalandcytochemical studies of the chromatoid body and related elements in the spermatogenesis of t:he rat. Quarterly Journal of Microscopical Science 102, 495-505.

Thiery, J.P. (1967j Mise Kevideneedespolysaccharides sur cou;;es fines enmicroscopie electronique. Journal de Microscopic 5, 987-1018.

Received: 29th October 1979 Accepted: 16th November 1979