morphological and histochemical studies of the chromatoid ...€¦ · in the spermatogenesi os f...
TRANSCRIPT
495
Morphological and Histochemical Studies of theChromatoid Body and Related Elements in the
Spermatogenesis of the Rat
By BHUPINDER N. SUD(From the Cytological Laboratory, Department of Zoology, University Museum, Oxford)
With three plates (figs. I to 3)
SUMMARY
In the spermatogenesis of the rat the chromatoid body is present during the growthof the primary and secondary spermatocytes, disappears at telophase of both themeiotic divisions, and is absent during interkinesis. It is reconstructed during the earlystages of spermateleosis but after the elongation and condensation of the nucleus itgradually becomes smaller and disappears. Simultaneously, in the caudal region vonEbner's stainable granules appear and gradually fuse together to form a single volumin-ous body, Regaud's sphere chromatophile, which is discarded with the residual bodyand is phagocytosed by the Sertoli cell.
The histochemical studies reveal that the chromatoid body, von Ebner's stainablegranules, and the sphere chromatophile are similar in composition. They consist mainlyof RNA and proteins, and this suggests that they may be centres of protein synthesis.The RNA content of von Ebner's stainable granules and the sphere chromatophileappears to be higher than that of the chromatoid body. This probably means that thereis a progressive decrease in the protein component of the chromatoid material. Alsothere is a distinct change in the chemical composition of the protein component of thechroma tin during the late stages of spermateleosis. It is tentatively suggested that thefunction of the chromatoid material may be to provide basic proteins for the finalmaturation of the chromatin of the late spermatid.
It appears that the chromatoid elements originate from the ground cytoplasm anddisappear by merging into the latter.
An enigmatic granular satellite has been found associated with the chromatoid body.It differs from the latter in its chemical composition.
INTRODUCTION
IN my recent papers on the chromatoid body in spermatogenesis I em-phasized that this cytoplasmic inclusion needs to be studied by histo-
chemical methods and electron microscopy in order to formulate criteria forits identification and to trace its origin and physiological significance (Sud,1961 b, c). In this connexion the histochemistry of the chromatoid body in thegrass-snake, Natrix natrix, has already been studied and a tentative suggestionhas been made about its function (Sud, 1961 a, b). It was considered desir-able to devote the next study to the histochemistry of the chromatoid body inthe spermatogenesis of the rat, the species in which it was first discovered andlater studied in detail by a large number of prominent workers.
The chromatoid body of the rat was first figured in the young spermatid byvon Brunn (1876), and later studied by several authors in fixed cells (Benda,1891; Moore, 1893, 1894; Niessing, 1897; Lenhossek, 1898; von Ebner,
[Quarterly Journal of Microscopical Science, Vol. 102, part 4, pp. 495-505, 1961.]
496 Sud—Chromatoid Body in the Rat
1899; Duesberg, 1908, 1909; Regaud, 1910; Allen, 1918; Ludford andGatenby, 1921; Daoust and Clermont, 1955), in living cells under phase con-trast (Austin and Sapsford, 1951), and by electron microscopy (Watson, 1952;Swift, 1956). Except for the remark by Daoust and Clermont (1955) that thechromatoid body in the rat spermatid is deeply stained by pyronin (whichsuggests that it contains RNA), no attempt has previously been made to studythe histochemistry of the chromatoid body in this species.
The present paper includes a report on a detailed histochemical study of thechromatoid body, its granular satellite, the stainable granules {die tingirbarenKornchen) of von Ebner (1888), and Regaud's (1901Z1) sphere chromatophile, inthe spermatogenesis of the rat. In addition observations have been recordedon the morphology and behaviour of these cytoplasmic inclusions and theresults compared with those of previous authors.
MATERIAL AND METHODS
The white rat, Rattus norvegicus albinus, was used for the present investiga-tion. Specimens of different ages were tried and it was found most convenientto work with rats weighing about 250 g. In these the testes seem to be mostactive and show practically all stages of sperm-formation, especially spermate-leosis.
The testes were dissected out rapidly, divided into small bits by a sharprazor or a pair of scissors, and the pieces directly transferred to variousfixatives. Paraffin wax, collodion, and gelatine were used as embedding mediafor different histochemical tests as detailed in the appendix (p. 504).
For deamination van Slyke's mixture, introduced into histochemistry byMonne and Slautterback (1950), was used. It consists of 2 volumes of con-centrated aqueous sodium nitrite solution, 1 volume of glacial acetic acid, and5 volumes of distilled water. The preparations were deaminated in the freshlyprepared mixture for 12 h at room temperature, washed thoroughly in water,and the deaminated and control preparations subjected to exactly the sametreatment.
Fresh material was studied under phase-contrast microscope in 0-9% saline,to every 100 ml of which 0-2 ml of 10% anhydrous calcium chloride had beenadded (Baker, 1944), or in an isotonic solution of Armour's bovine plasmaalbumin, fraction V, in 075% Baker's saline. Neutral red and Janus greenwere used supravitally. Both these vital dyes were used by diluting o-1 ml ofa 0-5% aqueous stock solution of the dye with 5 ml of the above-mentioned0-9% saline. A small piece of the testis tubule was immersed in the dilutedsolution for 5 to 10 min, teased, and studied by direct microscopy.
. . . . .. RESULTS
Morphological studiesThe chromatoid body in the spermatogenesis of the white rat, R. norvegicus
albinus, appears for the first time during the growth of the primary sperma-tocyte. It is a homogeneous, spherical body, measuring up to 0-9 fi in diameter
Sud—Chromatoid Body in the Rat 497
(fig. 1, A; 2, B, c; 3, B). In the large primary spermatocyte there are one tothree, generally two, chromatoid bodies, usually lying on opposite sides of thenucleus on or near the long axis of the cell (fig. 2, E). One of these is the mainchromatoid body already present in the earlier growth period of the cell, andthe remaining one or two are very small additional chromatoid bodies in theform of prominent granules which appear during the later growth of the cell.The chromatoid bodies usually lie at some distance from the nuclear membraneand have no topographic relationship with the idiozome.
During the first meiotic division the chromatoid bodies persist as far as thelate anaphase stage, but are absent during interkinesis. During the growth ofthe secondary spermatocyte the chromatoid body reappears as a single Homo-geneous sphere measuring up to 1 -2 /x in diameter. It often lies close againstthe nucleus. Once again it is visible up to late anaphase, but later it disappears.
In the spermatid the chromatoid body reappears during the initial stage ofacrosome-formation. At the start it lies on one side of the nucleus but later itshifts to its posterior end (fig. 1, B, E.) It may have an irregular outline butultimately it assumes a spherical form and measures up to i-8 /A in diameter.Before the centriole spins out the axial filament, a lighter and a darker portionmay be distinguished in the chromatoid body in preparations stained withbasic dyes, basic dye-lakes, and particularly after the acid haematein test(fig. 1, B), but afterwards it appears homogeneous even in such preparations(fig. 1, E; 2, A). Invariably the chromatoid body in the spermatid is accom-panied by a granular satellite which either lies in contact with it (fig. 1, B)or adjacent to it: in the latter case the gap between the two is bridged byfaintly stained material (fig. 1, E).
When the cytoplasm shifts behind the nucleus to form the tail-bladder, thechromatoid body is seen adjacent to the nucleus within the manchette beside theaxial filament. Generally at this stage the granular satellite separates itself fromthe chromatoid body and lies at a little distance from the latter (fig. 2, A, c).After the elongation and condensation of the spermatid nucleus, while thecytoplasm of the tail-bladder is spreading further back along the axial filament,the chromatoid body undergoes rapid diminution without changing its posi-tion (fig. 1, c; 2 E), and ultimately disappears. The satellite apparently becomesconfused with the centrioles.
Simultaneously with the disappearance of the chromatoid body, the stain-able granules of von Ebner (1888) appear in the form of numerous granulesfilling the caudal region of the spermatid. These granules flow together intoa single large spherical body called by Regaud (19016) the sphere chromatophile(fig. 1, D; 2, F). The latter gradually shifts to the front end of the head of thematuring spermatozoon and becomes frothy and voluminous, assuming anirregular contour (fig. 1, E; 2, G; 3, F). The cytoplasm containing this body isdetached from the spermatozoon to form the residual body. The residualbodies containing the sphere chromatophile form a zone between the sperma-tozoa and the spermatogenetic cells, and they remain in this position for sometime after the spermatozoa are shed. The residual bodies remain connected
498 Slid—Chromatoid Body in the Rat
with the Sertoli cells, and the sphere chromatophile enclosed in each of themappears to pass to the base of the Sertoli cell (fig. 1, E). Some of the residualbodies, however, are not phagocytosed but are left behind in the lumen, wherethey degenerate.
The chromatoid body in the young spermatid, when studied in livingmaterial under the phase-contrast microscope, gives a high phase-change andappears slightly darker than the idiozome (fig. 3, G). Neutral red and Janusgreen, used supravitally, leave it untouched.
Histochemical Studies
In the primary spermatocyte even the larger chromatoid body is small incomparison with the size of the cell and therefore it is not invariably seen insections. In the secondary spermatocyte it is slightly bigger than that of theprimary spermatocyte but the cell is not of common occurrence since the lifeof the secondary spermatocyte only amounts to about 2% of the spermato-genetic wave (Roosen-Runge and Giesel, 1950). In the young spermatid,however, the chromatoid body assumes the largest size; in addition the cell issmall, and the body therefore generally appears in sections. For this reason thechromatoid body at the young spermatid stage of the rat lends itself best tohistochemical study. At this stage it is possible to find it even in preparationsshowing negative results, while in the spermatocytes it is rather difficult tolocate it in such preparations and the conclusions in some cases had to bearrived at after studying a large number of serial sections. Von Ebner's stain-able granules and the sphere chromatophile are convenient for histochemicalstudies.
The tests listed in the appendix on page 504 suggest the absence oflipid (even in solid or masked state) from the chromatoid body of spermato-cytes and spermatids, the granular satellite of the chromatoid body of thelatter, the stainable granules of von Ebner, and the sphere chromatophile(figs. 1, A-E; 2, A). The intense basiphilia of these bodies (figs. 2, B-G; 3, A) is
FIG. I (plate). Sections of the testis of R. norvegicus albinus; acid haematein (AH) afterpyridine extraction.
A, large primary spermatocytes showing in focus AH-positive main chromatoid body (cb)adjacent to the nucleus («).
B, early spermatids, duplex chromatoid body indicated by arrows, AH-positive satellite (s)in contact with the AH-positive chromatoid body (cb) lying on the side of the nucleus (K) in oneof the spermatids.
c, maturing spermatozoa showing the gradual reduction in the size of the AH-positivechromatoid body (indicated by arrows) situated near the base of the head (h).
D, maturing spermatozoa showing AH-positive von Ebner's stainable granules (sg), andAH-positive sphere chromatophile (sc); chromatoid body indicated by an arrow in an earlyspermatid.
E, irregularly shaped AH-positive sphere chromatophile (sc) at the front end of the spermheads (h), the one indicated by an arrow has become frothy; some spheres chromatophiles beingphagocytosed by the Sertoli cell (st); chromatoid body in early spermatids indicated by arrows,in one spermatid it is seen inside the manchette (mt); satellite (s) seen adjacent to the chroma-toid body of one of the spermatids.
FIG. I
B. N. SUD
FIG. 2
B. N. SUD
Sud—Chromatoid Body in the Rat 499
due to RNA. The basiphilia of the satellite may possibly be due to highlypolymerized RNA, since it shows special affinity for methyl green in thepyronin / methyl green test, is negative to this test after treatment withribonuclease, and is Feulgen-negative. It may further be mentioned that thestainable granules, the sphire chromatophile, and the satellite stain more readilyby some basic dyes (crystal violet and methyl green) than the chromatoidbody. This fact suggests that the proportion of RNA in the former is muchhigher than in the latter. The basiphilia of the structures concerned may alsobe partly due to acidic groups in protein.
The results of staining with acid dyes (fig. 3, B-F) and the Sakaguchi reactionwith and without previous deamination suggest that the chromatoid body, thestainable granules, and the sphere chromatophile possess basic proteins (pro-tein-bound arginine) and proteins with basic groups other than amino-groups.Arginine appears to be most abundant in the chromatoid body, less so in stain-able granules, and almost absent from the sphere chromatophile. Histonesappear to be absent from these bodies and arginine therefore presumablyoccurs as a component of protamines. The satellite lacks basic proteins. Thereappears to be a small amount of tyrosine in the chromatoid body, the stainablegranules, and the sphere chromatophile. Tests for alkaline phosphatase, acidphosphatase, and calcium are negative for all the bodies concerned. Thusthe histochemical study suggests that the chromatoid body, von Ebner'sstainable granules, and Regaud's sphire chromatophile are, in general, similarin composition, consisting mainly of RNA and proteins. Further, there is aprogressive increase in the RNA component and a corresponding decrease inthe arginine component of the chromatoid material (chromatoid body, stain-able granules, and sphere chromatophile). It also appears that the arginine inthese bodies occurs as a component of protamines rather than histones. Prota-mines are known to replace the histones of chromatin during spermatogenesis.
FIG. 2 (plate). Sections of the testis of R. norvegicus albinus.A, formaldehyde/calcium followed by AH test; early spermatids with AH-positive chrom-
atoid body (indicated by arrows) at the posterior end of the nucleus (n); AH-positive satellite(s) has detached itself from the chromatoid body.
B, Sanfelice / crystal violet; large primary spermatocyte showing in focus the basiphil mainchromatoid body (cb) situated away from the nucleus (n).
C, Sanfelice / crystal violet; primary spermatocyte (at the top) with basiphil main chrom-atoid body (cb) and faintly stained idiozome (i) situated adjacent to the nucleus (n); earlyspermatids showing the chromatoid body (cb) near the posterior end of the nucleus (n);basiphil satellite (s) detached from the chromatoid body seen in one of the spermatids.
D, Sanfelice / crystal violet; the labelled spermatid on the left with the basiphil chromatoidbody (cb) enclosed by the manchette (mi); the labelled spermatid on the right with the faintlystained idiozome (t) situated posteriorly to the basiphil chromatoid body (cb).
E, Sanfelice / crystal violet; maturing spermatozoa with anterior part of the heads (h) em-bedded in the Sertoli cell (st) and reduced basiphil chromatoid body (cb) at the posterior endof the nucleus; large primary spermatocyte with the main and two additional basiphil chrom-atoid bodies (indicated by arrows) and faintly stained idiozome (i) lying near the nucleus (n).
F, Zenker / basic fuchsine; maturing spermatozoa showing basiphil von Ebner's stainablegranules (sg) and basiphil sphere chromatophile (sc).
G, Zenker / basic fuchsine; irregularly shaped basiphil sphere chromatophile (sc) situated atthe front end of the sperm heads (h); a frothy sphere chromatophile indicated by an arrow.
2421.4 L 1
500 Sud—Chromatoid Body in the Rat
In the rat, as in many other animals, the spermatid nucleus can be seen toundergo a distinct change in the chemical composition of its chromatin duringthe late stages of spermateleosis. The following methods were used to studythis change.
(a) Sections of material fixed in Clarke's (Carnoy's) fluid, when stained withmethylene blue, show the chromatin of the spermatid nuclei stained but thenuclei of the spermatozoa are untouched.
(b) Sections of material fixed in Tellyesniczky's fluid, postchromed, stainedwith Ehrlich's haematoxylin and then with safranine (Regaud, 1901a) showthe chromatin of spermatocytes and spermatids blue and that of sperma-tozoa red.
(c) Sections of material fixed in Clarke, extracted with hot trichloraceticacid in order to remove DNA, mordanted with aluminium sulphate, stainedwith haematein, and blued (Baker, technique not yet published) show thechromatin of spermatocytes and spermatids blue and that of spermatozoabrownish.
(d) Sections of the material fixed in Zenker for 3 h, deaminated with vanSlyke's reagent, and stained with azocarmine show the chromatin of spermato-cytes and spermatids untouched and that of the late spermatids and sperma-tozoa red.
The distinct change in the chemical composition of chromatin revealed bythese tests, the presence of RNA and basic proteins in the chromatoid material,and the gradual decrease in the protein component of the latter suggest thatthe function of the chromatoid material may be to provide basic proteins forthe final maturation of the chromatin of late spermatids.
DISCUSSION
The present investigation has removed all doubts about the presence of thechromatoid body in the primary spermatocyte and spermatid of the rat. It hasalso proved that the chromatoid body, von Ebner's stainable granules, and
FIG. 3 (plate). Sections of the testis of R. norvegicus albinus.A, Zenker; pyronin / methyl green; von Ebner's stainable granules (sg) and sphere chromato-
phile (sc) stained deep red by pyronin; sperm heads (h) blue and tails (t) red.B, Sanfelice / acid fuchsine; the labelled cell is a large primary spermatocyte showing the
acidophil main chromatoid body (cb) near the nucleus (n) and a slightly stained idiozome (i).C, Sanfelice / acid fuchsine; early spermatids, the chromatoid body (cb) is deeply stained but
the idiozome (i) is faintly stained.D, Zenker / azocarmine; the labelled spermatid showing acidophil chromatoid body (cb) at
the posterior end of the nucleus («).E, deamination / azocarmine; early spermatids showing acidophil chromatoid body (labelled
cb or indicated by an arrow), completely negative idiozome (i) and chromatin; maturing spermheads (k) are deeply stained.
F, Zenker / azocarmine; acidophil sphere chromatophile (sc) lying alongside the maturingsperm heads (h) or in front of the latter.
G, living cells under phase contrast mounted in a slightly hypertonic protein medium witha refractive index of 1'354; the labelled cell is an early spermatid showing the chromatoid body(cb) in contact with its satellite (s) lying near the posterior end of the nucleus (n), and idiozome(i) at the anterior end of the latter showing acrosome-formation.
Sud—Chromatoid Body in the Rat 501
Regaud's sphere chromatophile are similar in composition, consisting mainly ofRNA and proteins with basic groups. The chromatoid body of the snake(Sud, 1961 a, b) has a similar chemical composition.
It has been suggested that the chromatoid body of the rat arises from thenucleus (Benda, 1891; Moore, 1893, 1894; Regaud, 1910) or nucleolus (Len-hossek, 1898; von Ebner, 1899; Fischer, 1899; Duesberg, 1908). von Ebner(1888) states that his stainable granules cannot arise from the nucleus sincethey first appear when the head of the spermatozoon has taken on its finalform. I am inclined to think that the chromatoid body and von Ebner'sstainable granules originate from the ground cytoplasm, as suggested for thechromatoid body of reptiles (Sud, 1955, 1956, 1957).
As to the fate of the chromatoid body in the rat, a claim has been made thatit is absorbed into the nucleus (Moore, 1893, 1894). Other suggestions are thatit gradually becomes smaller during the final stages of spermateleosis and thenfragments into two or three pieces, which dissolve in turn insensibly into thecytoplasm (Duesberg, 1909), or that it is sloughed off with the residual cyto-plasm (Austin and Sapsford, 1951). I believe that during the final stages ofspermateleosis the chromatoid body gradually merges into the ground cyto-plasm and simultaneously von Ebner's stainable granules arise from theground cytoplasm in the caudal region and run together to form a singlesphere chromatophile, which is cast off with the residual body. Some of theresidual bodies are phagocytosed by the Sertoli cells while others degeneratein the lumen. According to von Ebner (1888) the stainable granules arederived from the Sertoli cells and they go back into the Sertoli cells. Lacy(i960), however, believes that the RNA of the residual bodies is formed by thegerm cells and later enters the Sertoli cells, where it is absorbed or dispersed.
Gresson and Zlotnik (1945) use the term 'accessory bodies' for a cytoplasmicinclusion in spermatogenesis of the rat which they consider to be the same aswhat others have called chromatoid bodies. According to them the 'accessorybodies' arise from the localized Golgi material. They describe their accessorybodies as argentophil and stainable by iron haematoxylin and acid fuchsine.This supposed homology has already been opposed by myself (Sud, 1961c).I have suggested that the 'residual body' described by Gresson and Zlotnik inthe spermatid of this species is in fact the chromatoid body. The present in-vestigation supports my earlier opinion.
The granular satellite found in association with the chromatoid body of thespermatid in the present investigation has been shown to differ in its chemicalcomposition from the chromatoid body. The evidence suggests that it consistsof highly polymerized RNA. The satellite may be represented by the lamellararrays that are seen to be associated with the chromatoid body in electronmicrographs of the rat spermatid by Swift (1956). It is also just possible thatthe satellite, after detachment from the chromatoid body, forms the end-knobor basal body of the axial filament, since, like the satellite, the end knob is alsostained blue in pyronin / methyl green preparations and both are of approxi-mately the same size.
502 Sud—Chromatoid Body in the Rat
I take this opportunity for expressing my gratitude to Dr. J. R. Baker,F.R.S., for his kind supervision and helpful discussions, and for translatingGerman and French papers. I am grateful to Professor Sir A. C. Hardy,F.R.S., for providing research facilities in his Department, and to the Vice-Chancellor of the Panjab (India) University for granting me study-leave. Iwish to thank Mrs. B. M. Luke and Mr. P. L. Small for their valuabletechnical help.
REFERENCES
ALFERT, M., and GESCHWIND, I. I., 1953. Proc. Nat. Acad. Sci. Wash., 39, 991.ALLEN, E., 1918. J. Morph., 31, 133.AOYAMA, F., 1929. Z. wiss. Mikr., 46, 489.AUSTIN, C. R., and SAPSFORD, C. S., 1951. J. roy. micr. Soc, 71, 397.BAKER, J. R., 1944. Quart. J. micr. Sci., 85, 1.
1946. Ibid., 87, 441.1947- Ibid., 88, 115.1949- Ibid., 90, 293.1956a. Ibid., 97, 621.19566. Ibid., 161.!959- J- roy. micr. Soc, 77, 116.and JORDAN, B. M., 1953. Quart. J. micr. Sci., 94, 237.
BENDA, C, 1891. Arch. f. Physiol., physiol. Abt. (no vol. number), 549.BRADBURY, S., 1956. Quart. J. micr. Sci., 97, 30a.
and CLAYTON, B.-P., 1958. Nature, 181, 1347.BRUNN, A. v., 1876. Arch. mikr. Anat., 12, 528.CHAYEN, J., GAHAN, P. B., and LA COUR, L. F., 1959. Quart. J. micr. Sci., 100, 379.DANIELLI, J. F., 1946. J. exp. Biol., 22, no .
1947. Symp. Soc. exp. Biol., 1, 101.1950. Cold Spring, Harb. Symp. quant. Biol., 14, 32.
DAOUST, R., and CLERMONT, Y., 1955. Amer. J. Anat., 96, 255.DUESBERG, J., 1908. Arch. Zellforsch., 1, 399.
1909. Ibid., 2, 137.EBNER, V. v., 1888. Arch. mikr. Anat., 31, 236.
1899. S.B. d. k. Akad. Wiss. math.-naturw. CL, Abt. 3, 108, 429.FERNSTROM, R. C, 1958. Stain Tech., 33, 175.FEULGEN, R., and ROSSENBECK, H., 1924. Z. physiol. Chem., 135, 203.FISCHER, A., 1899. Fixvrung, Farbung und Bau des Protoplasmas. Jena (Fischer).GATENBY, J. B., and BEAMS, H. W., 1950. The microtomist'sv ade-mecum (Bolles Lee). London
(Churchill).GOMORI, G., 1952. Microscopic histochemistry. Chicago (University Press).GRESSON, R. A. R., and ZLOTNIK, I, 1945. Proc. roy. Soc. Edinb., B, 62, 137.JORDAN, B. M., and BAKER, J. R., 1955. Quart. J. micr. Sci., 96, 177.LACY, D., i960. J. roy. micr. Soc, 79, 209.LENHOSSEK, M. v. 1898. Arch. mikr. Anat., 51, 215.LEWITSKY, G. A., 1931. Bull appl. Bot. Genet. Plant Breeding, 27, 175.LISON, L., 1953. Histochimie et cytochimie animates. Paris (Gauthier-Villars).LUDFORD, R. J., and GATENBY, J. B., 1921. Proc. roy. Soc. B, 92, 235.MCMANUS, J. F. A., 1948. Stain Tech., 23, 99.MONNE, L., and SLAUTTERBACK, D. B., 1950. Ark. Zool., 1, 455.MOORE, J. E. S., 1893. Anat. Anz., 8, 683.
1894. Int. Mschr. Anat. Physiol., I I , 129.NIESSING, C, 1897. Arch, mikr., Anat., 48, i n .PEARSE, A. G. E., 1954. Histochemistry, theoretical and applied. London (Churchill).PRZEIECKA, A., 1959. Quart. J. micr. Sci., 100, 231.REGAUD, C, 1901a. Arch. Anat. micr., 4, 101.
19016. Ibid., 231.
Sud—Chromatoid Body in the Rat 503
REGAUD, C , 1910. Arch. Anat. micr., n , 291.ROSSEN-RUNGE, E., and GIESEL, L., 1950. Amer. J. Anat., 87, 1.SUD, B. N., 1955. Res. Bull. Panjab Univ. India, 75, IOI.
1956. Ibid., 86, 31.1957. Ibid., 116, 345.1961a. Biochem. J., 78, 16P.19616. Quart. J. micr. Sci., 102, 51.1961c. Ibid., 273.
SWIFT, H., 1956. J. biophys. biochem. Cytol., 2, No. 4, Suppl., 415.WATSON, M. L., 1952. Univ. Rochester Atomic Energy Project. Atomic Energy Report,
UR-185, unclassified.WEIGL, R., 1910. Bull. int. Acad. Sci. Cracovie, B (no vol. number), 691.
APP
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DIX
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(co
nt.)
Test
s ap
plie
d
Test
tolu
idin
e bl
ue f
or
met
achr
omas
yth
ioni
ne f
or
met
achr
omas
ype
riod
ic a
cid
/ Sc
hiff
Feul
gen
pyro
nin
/ m
ethy
l gr
een
P/M
G a
fter
tri
chlo
race
tic
acid
P/M
G a
fter
ri
bonu
clea
seba
sic
fuch
sine
(0
-5%
aq
.;
RT
) fo
rba
siph
ilia
safr
anin
e (0
-5%
aq
.; R
T)
for
basi
phil
iasa
fran
ine
(0-5
% a
q.;
RT
) af
ter
tric
hlor
-ac
etic
aci
dN
ewto
n's
crys
tal
viol
et
New
ton'
s cr
ysta
l vi
olet
(s
tain
ing
at
5O°C
)N
ewto
n's
crys
tal
viol
et
New
ton'
s cr
ysta
l vi
olet
af
ter
tric
hlor
-ac
etic
aci
dC
resy
l vi
olet
as
used
fo
r N
issl
bod
ies
met
hyl g
reen
(1%
aq
.; R
T)
for
basi
phil
iam
ethy
l gre
en (
1% a
q; 5
O°C
) for
bas
iphi
lia
iron
hae
mat
oxyl
inE
hrli
ch's
ha
emat
oxyl
in /
saf
rani
ne(Z
waa
rdem
aker
's)
Fix
atio
n
ZZ
; C
lZ
Z;
Cl
Z 3
h
Z 3
h
Z 3
hZ
3 h
Z 3
h;
LS
Z 3
h
Z 3
h;
LS
; C
l
Z 3
h s sZ
3 h
Z3
hZ
3 h
Z 3
hT
z4
h
Em
bed-
ding
med
ium
P P P P P P P P P P P P P P P P P P P
Thic
k-ne
ss o
fse
ctio
nsin
fj.
10
10 8 6;
8
7; .
6
7; 1
6
7; 1
67 6 6 6 6 16 16 6;
16
7 7 7 6
Ref
eren
ce
Bak
er
(unp
ubli
shed
)B
aker
(u
npub
lish
ed)
McM
anus
, 19
48;
Pear
se,
1954
Feul
gen
and
Ros
sen-
beck
, 19
24Jo
rdan
and
Bak
er,
1955
Pear
se,
1954
; Jor
dan
and
Bak
er,
1955
Bra
dbur
y, 1
956
— —
Pear
se,
1954
Bak
er
an
d J
orda
n,19
53B
aker
an
d J
orda
n,19
53B
aker
an
d J
orda
n,•9
53B
aker
an
d J
orda
n,19
53;
Pear
ce, 1
954
Fern
stro
m, 1
958
— — —R
egau
d, 1
901a
Chr
o
Insp
erm
a-to
cyte
s
O OO
to
+
O
+ +
+(r
ed)
+ 4-
(red
)O
4. 4
-4-
4-4-
4-O O O
4-4-
4-
O 4-4- O
4-4-
4-4-
4-4-
4-
Res
ults
mat
oid
body
In
sper
mat
ids
O OO
to
+
O
4-4-
4-(r
ed)
4- 4
-(r
ed)
O4.4
.4-
4-4
-4-
O O O
+ +
+O 4- 4
-O
4-4-
4-4-
4-4-
4-
ibta
ined
•von
Ebn
er's
stai
nabl
egr
anul
es
O 4. 4
.O
to
+
O
4-4-
4-(r
ed)
+ to
+ +
(red
)O
4.4
.4-
4-4.
4-O
Oto
+
+ +
+
+ +
+O
4-4
.4.
4-4
-4-
4-4
-4-
4-4
-4-
4-4
-4-
Reg
aud'
ssp
here
chro
m-
atop
hile
O4-
4-O
to
+
O
4. 4
- +
(red
)
(red
)O
4-4
-4-
4-4
-4-
O
O t
o +
+ +
+4-
+ +
O
4-4
-4.
4-4
-4-
4-4
-4-
+ +
+4-4
-4-
Sate
llite
O — O O
4-4-
4-(b
lue)
4- 4
-(r
ed)
O4-
4-4-
4-4-
4-O
+ +
+
+ +
++
4-4
-
O — — —4-
4-
+—
APP
EN
DIX
{co
nt.)
Tes
ts a
ppli
ed
Tes
t
acid
fu
chsi
ne
(i%
aq.
; R
T)
for
acid
-op
hilia
eosi
n (i
% a
q.;
RT
) fo
r ac
idop
hilia
xylid
ine
red
(01
% i
n i%
ac
etic
aci
d;R
T)
azoc
arm
ine
(2%
aq.
, ac
idif
ied;
50°
C)
deam
inat
ion
by v
an S
lyke
rea
gent
/ a
zo-
carm
ine
(2%
aq.
, ac
idif
ied;
500 C
)Sa
kagu
chi
deam
inat
ion
by v
an
Slyk
e re
agen
t /
Saka
guch
i
Hg/
nitr
ite
coup
led
tetr
azon
ium
alka
line
fast
gre
en m
etho
d
Gom
ori's
for
alk
alin
e ph
osph
atas
ele
ad m
etho
d fo
r ac
id p
hosp
hata
seco
balt
met
hod
for
calc
ium
Fix
atio
n S
Z3
hZ
3h
Z3
h
Z3
h
z z
F/S
; A
leF
; A
le
F
Alc
/Ace
tA
cet
F/M
/P
Em
bed-
ding
med
ium
P P P P P P P
C,
PC
PP,
PC
P
P, P
CP
P P P
Thi
ck-
ness
of
sect
ions
in n
6 7 7 7 7 16 16 26 6, 1
0
6, 1
0
8 81
0
Ref
eren
ce
— —
Gat
enby
and
Bea
ms,
195°
Mon
ne a
nd S
laut
ter-
back
, 19
50B
aker
, 19
47M
onne
and
Sla
utte
r-ba
ck,
1950
, B
aker
,19
47B
aker
, 19
566
Dan
ielli
, 19
47,
1950
;Pe
arse
, 19
54A
lfer
t an
d G
esch
-w
ind
ioci
Gom
ori,
1952
Gom
ori,
1952
As
in a
lkal
ine
phos
-ph
atas
e te
st
used
by D
anie
lli,
1946
Res
ults
obt
aine
d
Chr
omat
oid
body
Insp
erm
a-to
cyte
s
+ +
+
— —
+ +
+
+ +
+-J-
+ O
O t
o +
— O O O
In s
perm
atid
s
+ +
+
+ +
++
+
+ +
+
+ +
+
O + O O O O
von
Ebn
er's
stai
nabl
egr
anul
es
+ +
+
+ +
++
+
+ +
+
+ +
+
O
O t
o +
-t- O O O O
Reg
aud'
ssp
here
chro
m-
atop
hile
+ +
+
+ +
+
+ +
+
+ +
+O
to
+O + + O O O O
Sate
llit
e
O O O O O O O O O O O O O
Key
. A
le =
al
coho
l; A
cet
=
acet
one;
Alc
/Ace
t =
ab
solu
te a
lcoh
ol a
nd a
ceto
ne i
n eq
ual
volu
mes
; A
oy =
A
oyam
a's
flui
d; a
q. =
aq
ueou
s; C
=
coll
odio
n;C
l =
C
lark
e's
(Car
noy'
s)
flui
d;
Cr/
F =
ch
rom
ium
tri
oxid
e/fo
rmal
dehy
de;
F =
fo
rmal
dehy
de;
F/C
a =
fo
rmal
dehy
de/c
alci
um;
Flm
=
Fle
mm
ing'
s fl
uid;
F/M
/P =
fo
rmal
dehy
de/m
etha
nol/
pyri
dine
; F
/S =
fo
rmal
dehy
de/s
alin
e;
G =
gel
atin
e;
LS
=
Lew
itsk
y-sa
line
; P
=
para
ffin
; P
C =
po
st-c
hrom
ed;
PC
P =
Pe
terf
i's
cello
idin
-par
affi
n m
etho
d;
PO =
po
stos
mic
ated
; R
T =
roo
m t
empe
ratu
re;
S =
Sa
nfel
ice'
s fl
uid;
T
=
T
elly
esni
czky
's f
luid
; W
B +
PE
=
wea
k B
ouin
+ p
yrid
ine
extr
acti
on;
Z =
Zen
ker's
fl
uid;
3
h =
3
hour
s;
+-)
--!-
= s
tron
g re
acti
on;
+ +
=
m
oder
ate
reac
tion
; +
=
w
eak
reac
tion
; O
=
no
to I a"