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The Ultrastructure of Human Testicular Tumors. I. Interstitial Cell Tumors
THE JOURNAL OF UROLOGY
Vol. 93, No. 1 January 1965 Copyright© 1965 by The Williams & Wilkins Co. Printed 1:n [l.S.A.
THE ULTRASTRUCTURE OF HUl\IAN TESTICULAR TU:\IORS. I. INTERSTITIAL CELL TU~\IORS THEODOTrn F. BEALS,* G. BAH.RY PIERCE, JR.t
A0!D
C. F. SCHROEDER
Fram the Department of Pathology, The University of Michigan Medical Center, Ann Arbor; ancl the Wyandollc General Hospital, Wyandotte, ilf-ichiga:n
ination of a 23-year-old University student; surgically it proved to be a mass in the upper pole of the left testis 1neasuring 2 by 1.5 by 1 cm.. * It was yellowish in color, lobulated, and encapsulated. Light microscopically the tumor cells were polygonal in shape with oval, centrally placed nuclei and intensely eosinophilic cytoplasm with many pigmented granules (fig. 1). Sorn.c sections of the tumor had an organoid appearnnce with cords and clumps of Leydig cells separated by vascular spaces, while in others the cells were spread diffusely. Spindle-shaped interstitial cells described by Sniffen8 were not observed. N umerous crystalloids of Reinke were scattered in a patchy manner throughout the tumor. The crystalloids were slender, eosinophilic, homogeneous bars measuring 2 to 3 microns in diameter by 30 to 40 microns in length. A space which was probably a fixation artifact often surrounded the crystalloids (fig. 2). l\!Iarked atrophy of the interstitial cells of the adjacent normal testicular tissue was observed. The other interstitial cell tumor (M-31) was obtained surgically from the left testis of a 7-yearold boy who presented with a classical virilizing syndrome. Preoperatively, the level of 17-ketosteroids was 7.2 mg. per 24 hours. The tumor was encapsulated, 2 cm. in diameter, soft, goldenbrown in color, and exceedingly vascular. It was surrounded by a thin rirn of compressed testicular tissue. Microscopically cords of Leydig cells separated by vascular sinusoids imparted an organoid pattern to the tumor (fig. 3). Crystalloids of Reinke were not observed. This patient's endocrinopathy was terminated with removal of the tumor; postoperatively the level of 17-ketosteroids was 1.5 mg. per 24 hours. For comparison, interstitial cells were obtained from normal testicular tissues obtained at orchiectomy of young men with testicular tumors. In view of the pressure effects of the tumors and the frequency of associated endocrine anomalies,
Tumors of the interstitial cells of Leydig have been associated with virilism in prepubertal patients and have led to the concept that interstitial cells of the testis secrete testosterone. Although several ultrastructural studies of the interstitial cells of lower species have been published,1·4 the descriptiorn of human interstitial cells by Fawcett and Durgos 5 and Yamadan are the only ones available. No interstitial cell tumors have been described electron microscopically. During investigations upon the pathogenesis of human testicular tumors,7 we have studied two interstitial cell tumorn ultrastrueturally and compared the findings to those of normal and hyperplastic Leydig cells. MATERIALS AND ME'l'HODS
The first interstitial c,ell tumor (M-9) of this study was discovered on routine physical examAccepted for publication .June 15, 1964. Supported in part by grants from the National Institutes of Health (CA-G113) and the American Cancer Society, Inc. (E-105). * Medical Student Hescarch Fellow. t Markle Scholar in Medical Science. 1 Carr, I. and Carr, .J.: Membranous whorls in the testicular interstitial cells. Anat. Rec., 144: 143-147, 19G2. 2 Christensen, A. K. and Fawcett, D. W.: The normal fine structure of opossum testicular interstitial cells . .J. Biophys. Cytol., 9: G53-G70, 19Gl. 3 Leeson, C. H.: The fine structure of rat interstitial tissue. In: Proceedings of the Fifth International Congress for Electron Microscopy. Edited by S.S. Breese, .Jr. New York: Academic Press, 1962, vol. 2. 4 Yasutaki, S.: Fine structure of the mouse testicular interstitial ceJI. In: Proceedings of the Fifth International Congress for Electron Microscopy. Edited by S.S. Breese, Jr. New York: Academic Press, 1962, vol. 2. 5 Fawcett, D. W. and Burgos, M. H.: Studies on the fine structure of the mammalian testis. IL The human interstitial tissue. Amer . .J. Anat., 107: 245-269, 1960. 6 Yamada, E.: Some observation on the fine structure of the interstitial cell in the human testis. In: Proceedings of the Fifth International Congress for Electron Microscopy. Edited by S. S. Breese, Jr. New York: Academic Press, 19G2, vol. 2. 7 Pierce, G. B., Jr.: The pathogenesis of testicular tumors. J. Urol., 88: 573-584, 1962.
Specimen contributed by R. M. Nesbit, M.D. Sniffen, R. C.: The testis. I. The normal testis. Arch. Path., 50: 259-284, 1950. *
8
G4
UJ/[R.\STR11CTURJ<1 OF H\iJ\lAi'- TESTICULAR TUJ\IOllci
(\:j
ethyl alcohols, and embedclerl in \'estopttl \\' using (.he method of Kurtz. 10 Sections 11·cn• cut on a Portp1·-Blum micmtome and stained eiiher \\it.h aqm'OlL, uranyl acetate for 24- honr., m l pl:l' cen ( lead acdatP in mctlmuol for ;-30 minute.'i. Ub'ii'n·a"liorrn 11-cre n1.ack on an RC"\. EMl--:iF del'tron micro~cope. rn:S.ULTS
1·ariatio1Js [mm extreme hypolcydigism to marked degrees of interstitial hyperplasia ,n,re anticiand encountered in thPse organs. For the ultra.st.rudural studirs, the tis,,ues were placed immediately in cold , eronal buffered osmium tetroxic!e 1Yith sucrose, 9 and minced. After l hour they were dehydrated in graded
l'lirn'it.ructurn1ly the cp]h, of tlm'iC' tumor,, \\TJ'f; similar to eacl1 ot.lwr and to nonnal inwr~lit ial cdb (fig'i. 4, 5 and (-i ,B). \Yhcrra, tlw 1rn111cl nm:lri ol' the: polygonal cclb wen· not remarkable . thP cytoplasm \Yas characterized Ow Pxti'emb development of me111hrano11.s organ('llar 8\-'it.elll'i. In many cells, ,-csicular profiles of endopla,m1ie retieulnm ,,-ere ,-:o mm1e1·ous that the cy1oplasm re.sernbled the ~urfa,cc of a tine sponge. Among the'iP ,·Psicles ,n-n· scattered mitochomlria, 1·arious osmiophilic inclnsio11,, and occasional crystalloicb of Reinke. The cells of '\1-31 (figs . .5,.-1. and G), lbe ,-irilizing tumor, 1n:re indistinguishable frnm thu.s(· of normal interstitial cells (fig. 4). The majol'ilY of eel],; of l\I-31 contained myi iads of \'(\,id<"·' ,,imila1· to those describr:cl in the interstitial cdb of llH, opossum by Christensen and Fawcett." "\.hliough t.hcsc ,·esicles had a fe11· attached ribosome.,, the,· ,,-ill be rpferred to as agranular rndopla.smie retin1lum. It is not certain whether the.,· l'epre,en\ cli,wrete n'sicle'i 01· a system of anas1omosing tubule.c; as suggested by LePson. 3 ln some C<'lls, 1d1ich contained fe,wr n•sicles and thcrcbr gan' the general impre.,sion of being less well diff('t·e11 tiated thall thr others, lamellar forms of the endoplasmic reticulum were found (fi,g. 7). Th('se profiles were: few in number and like the Ye.c,icuiar component had only a ,catt.ering of att.ar·herl ribrnmnws. Typical granular enclopla,mic 1elirnlum was rarr and eonsistcd of seat.t<·t·erl ,hmt, prnfilcs (figs. 5, B and The rnosl rlrarnaCic nwmhranous configurations were whorls 7 and 8), which were identical to those de.scribed in tJw inter,;titial cells of mice by Carr and C,1n 1 ancl by Yasutaki. 4 Ther varied in size ttrn! complexity; the simplest were composed of only une m two ronccntric, ~parscly granular, rncmbranott~ ring, (fig. 8, B), 11·hcrea, the largest wrre rnmposed of as rnany as 30 agranular rings. The~r: rings ,surrnunded a niclus, u~ually eiiJH·r a mii,0-
'Caulfield, J.B.: Effects of varying the vehicle for OsO., in tissue fixation. J. Bioph 0·s. Cytol., 3: 827-830, Hl57.
10 Kurtz, 8. AL: A nnw method for 1,mbeddinµ: tissues in \'estopal W. ,J. lJltrastruct. Hes, 5: -,m-: l(i9' HJ(i 1.
!,'10. 1. ReprnsC'ntat.ivc portion of l\f-ll, inter-..ititial eel! Lumm, illustrntes clillusc pattern of µ:rn\Tth. x:iOO.
Pru. 2 Cryst.alloicl of lfoinke surrounded ln· clear zone may be seen a1 arrow. :11-f-\J, interstiti,il cr ll wmor. x·480. 0
.Frn. 3. Organoid pattern typical of 1\l-31. X300
:1
66
BEALS, PIERCE, JR. AND SCHROEDER
Fm. 4. A, cytoplasm typical of interstitial cell from normal adult testis. Abundant branching vesicular endoplasmic reticulum forms background for mitochondria (m) and angular inclusions (a). X 18,600. B, portions of 2 interstitial cells from normal adult testis. Yesicular endoplasmic reticulum is seen as oval profiles dispersed throughout cytoplasm. n-nucleus; L-lipid droplets distorted by preparative procedures. X14,500.
T_;LTRASTRLTCTURE OF 1-rc:\IAN TESTJCULAR TL:\IORS
chonclrion or a densl: osmophilie; granule. At the of Lhe 1YJ:10rl the rnmponent membram:s appeared contimwus 1-rith the H'sicular and lamellar form~ of the endoplasmic reticulnm (fig. 8: A) In general, celb in which whorls 1Yerc obseiTed WPt·r poorly differentiated. Vifhorls wen, obc,ern-~d in lVl-31 and normal internCitial cells, but 1Yen: frequent components of the cytoplasm of ::\l-\l. Althougb prnfiles oi' the Golgi apparatL1s were seen in all ~pccimen~, tbey were oYershadowed by the Yesicular component of the cytoplasm in l\T31 and in the nornrnl interstitial Cl'lk Mito-· chondria \Ycre numerous and pkomorpl1ic, wiU1 moderately electron dense matrices that made them. appear dark in relation to their surroundings 5, 7 ancl ) . For the most pal't the cristae had the usual configuration althongh in some imtances appeared tubular (figs. 6,A. and 9,A). Of the numernus inclusiorn, obsern,cl in the~e cells, the mo~t oufatancling were Che cry.~talloiJs of Rcinke, 11 the ultrastructure of whif:h was :first described Fawcett and Bmgos. 5 They ,n:re angular in shape, not enclosed by memand their internal arrangement was that of a fine lattice. Seen in one plane, the subunits
as lon?;, thin fib rib (fig;. 9,B), while in other planes as more or less discrete symmetrically spaced, den.,c particles (fig. 9,A). i\.lthough typically only a large single crystalloid was seen in the cytoplasm, occasional cells c:ont,ained numerous smaller ones. ,\ few were observed lying free in the intracellular space. The crystalloids m formalin-fixed 11ost-osmicated t,issue were smroundecl by ntcuolated zones JO) 11-h1ch are presumably responsible for the clear space observed around crystalloids when prepared for light microscopy (fig. 2). Other inclusion~ ,aried considerably in appearance. There were ~mooth membrane-enclosed on1l or round, densely osmiophilic masses (figs. 5,B and 6,B), complex strnctnres with pleomorphic componentf (fig. 6,B), mm1crous lipid in~lusions (fig~. 4,B and 5,,1), and inclusions which were descrilied by Fawcett and Bmgos" as lipochrome pigment granules. Filamentous fascicules, .-lescribed by Fawcett and and Yamacla 6 in normal Leydig cells were not observed in the tumors. Dense, sometimes angular, inclusions often Been in tlw c~-topla.~m of cell~ containing Reinke, .F Beitrii.ge zur histologie des meuschen. Arch. f. mikr. Anut, 47: :,4--4.4, 189fi.
crystalloids of Heinke 4,--1 nnrl nw.y be precursors of c:t·ystalloids. It should be repeated that the r:elJ., nf i\L.H and the normal cells \\'NC 80 mu"h aJik(, that they could only be distiugui,,iwd thr: absence of crystaJloids in the funner the other hand, contained many whorl3, fewer profiles reticulum, and in general gave the being ll'SS ,wll-cliffercntiat.ccl than either ;\J :JJ or the normal Leydig celh DISCUSSJO.N.
electron micro,,copic st udit','-' nf I.um.ors were made in the hope 1hat consislent ulrrastructural differences might be obsen·cd betwceu neoplastic and nmmal cells that \\'ould l<'ad t,o 11 better unclerntanding of the neopla,I ic 'ik1.te. These hopes 11-ere unfu1fil1ed; in ract t-wncir, oft.en retained enough of the 1111:rastructural featun'8 of their normal counterparts to a11on establisl1rnent of histogenesis. 12 io The celb of the 2 11trnors examined in this study arc almost exan co1rnterparts of normal Leydig cells. In addition to findings 1.·eportc:d by Fa11ceU and Burgos 5 fo1· normal human inter~tit,ial whorb identical to those described Carr and 1 Carr in Lhe interstitial cells of miee lrnni bt)Pn obsern,d in the tumor~ and in normal interstitial cells. The presence of attached ribosomes (admittedly few in number) and the of the peripheral membrane,, of 1lwsc: whorls ,Yith the endoplasmic: reticulum, lead us to be· lieve that the whorl is composed of profiles uf endoplasmic reticulum ancl is, therefore, analogous to the nebenkern of tho pituitary adenornas dc:sc:rihecl b3- Oberling and Bernharcl.rn Although it is admitted that intcrpretatwn
or
1 2 Zelickson, A. S.: Electron of the basal cell epitheliorna. J.
S\,Utiy-
[)errn'.,
39: 18:3-187, HJG2. 13 Pierce, G. B., Jr. and Midgley, ,\.. H., ,Jr.· Origin and function of humm1 syncytiot rophoblastic giant cells. Amer. J. Path 43: 15~l-173,
19(i3 H Pierce, G. B., Jr., Midgley, A. ll., ,Jr., H.mn, ,). S. and Feldman, J. D.: Parietal yolk cnrcinoma: Clue 1,o the histogenesis of 'R membrane of 1.he mouse embryo. Arner. ,J. Path.
41: 549-56G, 19Ci2 . 15 Dalton, A. J., Law, L. W., J\folorn,y. J. 13. ;;.ml :VIanaker, R. A.: All electron microscopic ol a series of murine lymphoid neoplasms. J. ;,,1;. Cancer Inst., 27: 747-791, 19Gl. 16 Oberling, C. and Bernhard, W.: The morphology of the cancer cells. In: The Cell i½ed Cells, Part 2. Editerl by J. Braclrni Mirsky. Xew York: Academic Press, 19Gl, vol.+.
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F.rn. G. .i1. k,ss welJ-differnntiated of cell from 1[-31 shows portion of whorl (w) ancl mitochondria (m) with both larnellar and cristae. )(2:i,500. B, portion of neoplastic cell from !II-:ll which did not contain whorls and wbich has nrnny incltrnious and vesicles in its cytoplasm. XlS./500.
FrG. 5. A cytoplasm of neoplastic ('Pll representative of those in :\1-:31, virilizing inlerntitial cell tumor from of 7-year-olci ; Golgi bodies (g) blend with numerous profiles of vesinrbr reticulum. IVfany inclusions caa seen scattered through ; plcomorphic densf: body brnne-endosed masses (arrow J. and mitochonclrin (m). X lG,000 B, cytopla.srn of 2 adjacent cells from 11-\J interntitial cell tumor from testis of less well-diffenmtiatccl than M
70
BEALS, PIERCE, JR. AND SCHROEDER
FIG. 7. Cells illustrating degree of differentiation characteristic of M-9; majority of membranes in this figure are portions of cellular membrane. Note lack of endoplasmic reticulum and clusters of Golgi profiles. N-nucleus; w-whorl. Xl0,500.
electron micrographs can lead to erroneous functional concepts, it is worth pointing out that M-31, a virilizing tumor, contained few whorls and many vesicles of endoplasmic reticulum. Cells with whorls contained less vesicular endoplasmic reticulum than those without whorls. These data might suggest that whorls are not necessary for steroid production and may represent an inactive storage arrangement of the endoplasmic reticulum of cells which have stopped synthesizing specialized products, in this case, steroids. Porter17 has pointed out the association of steroid production and a cytoplasm containing numerous vesicular elements of agranular endoplasmic reticulum. The crystalloids of Reinke which are considered to be specific for Leydig and ovarian hilus cells have occasioned considerable speculation. The 17 Porter, K. R.: The ground substance; observations from electron microscopy. The Cell. Cells and Their Component Parts. Edited by J. Brachet and A. E. Mirsky. New York: Academic Press, 1961, vol. 2.
lattice-like structure bears a remarkable similarity to the yolk platelets in amphibian oocytes18 and the intranuclear protein crystals associated with adenovirus infections of HeLa cells. 19 Their composition and functional role in human interstitial cells have not been determined; they could represent the storage form of a specific protein or a type of abnormal secretion-product of the cells. Interstitial cells and adrenal cortical rests which are often observed in the testis have rnany features in comrnon. Crystalloids, of course, are only observed in the former. Ultrastructural studies of the adrenal cOitex under a 18 Wallace, R. A. and Karasaki, S.: Studies on amphibian yolk. 2. The isolation of yolk platelets from the eggs of Rana pipiens. J. Cell Biol., 18: 153-166, 1963. 19 .Morgan, C., Godrnan, G. C., Breitenfeld, P. M. and Rose, H. M.: A correlative study by electron and light microscopy of the development of type 5 adenovirus. I. Electron microscopy. J. Exp. Med., 112: 373-382, 1960.
Fru. 8. A and B, whorls in cytoplasm of cells from M-9 demonstrate continuity of membranes whorl with vesiculttr and lamellar components of endoplasmic reticulum. Arrow points to dense inclusion surrounded by 2 concentric membranes which could be early stage in formation of whorl.. X Hl,000 and lG,000. 71
72
BEA.LS, PIERCE, JR. A.ND SCHROEDER
Fm. 9. A, highly magnified view of crystalloid of Reinke demonstrates regularly oriented components of crystalline lattice. Arrow points to cleavage fault within crystalloid. X35,000. B, crystalloid of Reinke (er) contained within cytoplasm of interstitial cell from normal adult testis. Note large angular inclusions (a) which may be precursors of crystalloids. X17,000. variety of trophic conditions by Nishikawa and associates, 2° Carr, 21 • 22 Schwarz and asso20 Nishikawa, l\lI., l\!Iurone, I. and Sato, T.: Electron microscopic investigations of the adrenal cortex. Endocrinology, 72: 197-209, 1963. 21 Carr, I.: The ultrastructure of the human adrenal cortex before and after stimulation with ACTH. J. Path. Bact., 81: 101-106, 1961. 22 Carr, I. A.: The electron microscopy of the adrenal cortex. In: Conference on The Human Adrenal Cortex. Edited by A. R. Currie, T. Symington and J. K. Grant. Baltimore: The Williams & Wilkins Co., 1962, pp. 21-25.
ciates, 23 and Ross 24 described vesicular endoplasmic reticulum as a major component of the cytoplasm. Although it is not possible to set 23 Schwarz, W., Merker, H. J. and Suchowsky, G.: Electron microscopic studies on the effects of ACTH and stress on the adrenal cortex in rats. Virchow Arch. Path. Anat., 335: 165-179, 1962. 24 Ross, M. H.: Electron microscopy of the human foetal adrenal cortex. In: Conference on The Human Adrenal Cortex. Edited by A. R. Currie, T. Symington and J. K. Grant. Baltimore: The Williams & Wilkins Co., 1962, pp. 558-569.
ULTRASTRUCTURE OF HUMAN TESTICULAR TUMORS
73
FIG. 10. Interstitial cell from adult testis which was fixed with formalin prior to fixation for electron microscopy with osmium tetroxide. Lattice-like character of crystalloids of Reinke (er) is not evident and crystalloids are surrounded by artefactual zones of vacuolation, which could be responsible for clear spaces often seen around crystalloids prepared for light microscopy by formalin fixation. X 10,000. absolute ultrastructural criteria that distinguish adrenal cortical cells from Leydig cells, the presence of numerous microvilli and the higher frequency of lipid droplets in adrenal cortical cells and the large amount of the vesicular endoplasmic reticulum in the interstitial cells aid in the identification. Recent reports have stressed the clinical problem of differentiating a virilizing interstitial cell tumor from a hyperplastic adrenal rest in the testis of a patient with an adrenogenital syndrome.25, 26 Attempts at characterizing the testicular lesion on the basiR of steroid chemistry have not been too fruitful since an interstitial tumor that had crystalloids of Reinke and secreted testosterone contained appreciable amounts of 11-beta-hydroxylase activity, 26 an 2 5 Glenn, J. F. and Boyce, W. H.: Adrenogenitalism with testicular adrenal rests simulating interstitial cell tumor. J. Urol., 89: 456-463, 1963. 26 Savard, K., Dorfman, R. I., Baggett, B., Fielding. L. L., Engel, L. L., McPherson, H. T., Lister, L. M., Johnson, D. S., Hamblen, E. C. and Engel, F. L.: Clinical, morphological, and biochemical studies of a virilizing tumor in the testis. J. Clin. Invest., 39: 534-553, 1960.
enzyme considered to be of adrenal cortical origin. Glenn and Boyce 25 have reported that the prompt drop in level of urinary androgen;, following cortisone administration is the best evidence that a testicular nodule is a hyperplastic adrenal rest. SUMMARY
Two interstitial cell tumors are described and their ultrastructure compared with that of normal Leydig cells. The cells of the tumors and normal interstitial tissue are characterized by a cytoplasm containing numerous \ esicular elements of endoplasmic reticulum, and a variety of inclusions. One of the tumors showed numerous crystalloids of Reinke. In the other crystalloids were not seen, but the cells were otherwise indistinguishable from those of normal Leydig cells. The authors acknowledge help and criticism of Reed M. Nesbit, M.D., and B. Naylor, M.D., as well as technical assistance of Miss Faith Pruchnicki, Miss Jessica Goodwin and Mr. Matthew Harden.
Vol. 93, No. 1 January 1965 Copyright© 1965 by The Williams & Wilkins Co. Printed 1:n [l.S.A.
THE ULTRASTRUCTURE OF HUl\IAN TESTICULAR TU:\IORS. I. INTERSTITIAL CELL TU~\IORS THEODOTrn F. BEALS,* G. BAH.RY PIERCE, JR.t
A0!D
C. F. SCHROEDER
Fram the Department of Pathology, The University of Michigan Medical Center, Ann Arbor; ancl the Wyandollc General Hospital, Wyandotte, ilf-ichiga:n
ination of a 23-year-old University student; surgically it proved to be a mass in the upper pole of the left testis 1neasuring 2 by 1.5 by 1 cm.. * It was yellowish in color, lobulated, and encapsulated. Light microscopically the tumor cells were polygonal in shape with oval, centrally placed nuclei and intensely eosinophilic cytoplasm with many pigmented granules (fig. 1). Sorn.c sections of the tumor had an organoid appearnnce with cords and clumps of Leydig cells separated by vascular spaces, while in others the cells were spread diffusely. Spindle-shaped interstitial cells described by Sniffen8 were not observed. N umerous crystalloids of Reinke were scattered in a patchy manner throughout the tumor. The crystalloids were slender, eosinophilic, homogeneous bars measuring 2 to 3 microns in diameter by 30 to 40 microns in length. A space which was probably a fixation artifact often surrounded the crystalloids (fig. 2). l\!Iarked atrophy of the interstitial cells of the adjacent normal testicular tissue was observed. The other interstitial cell tumor (M-31) was obtained surgically from the left testis of a 7-yearold boy who presented with a classical virilizing syndrome. Preoperatively, the level of 17-ketosteroids was 7.2 mg. per 24 hours. The tumor was encapsulated, 2 cm. in diameter, soft, goldenbrown in color, and exceedingly vascular. It was surrounded by a thin rirn of compressed testicular tissue. Microscopically cords of Leydig cells separated by vascular sinusoids imparted an organoid pattern to the tumor (fig. 3). Crystalloids of Reinke were not observed. This patient's endocrinopathy was terminated with removal of the tumor; postoperatively the level of 17-ketosteroids was 1.5 mg. per 24 hours. For comparison, interstitial cells were obtained from normal testicular tissues obtained at orchiectomy of young men with testicular tumors. In view of the pressure effects of the tumors and the frequency of associated endocrine anomalies,
Tumors of the interstitial cells of Leydig have been associated with virilism in prepubertal patients and have led to the concept that interstitial cells of the testis secrete testosterone. Although several ultrastructural studies of the interstitial cells of lower species have been published,1·4 the descriptiorn of human interstitial cells by Fawcett and Durgos 5 and Yamadan are the only ones available. No interstitial cell tumors have been described electron microscopically. During investigations upon the pathogenesis of human testicular tumors,7 we have studied two interstitial cell tumorn ultrastrueturally and compared the findings to those of normal and hyperplastic Leydig cells. MATERIALS AND ME'l'HODS
The first interstitial c,ell tumor (M-9) of this study was discovered on routine physical examAccepted for publication .June 15, 1964. Supported in part by grants from the National Institutes of Health (CA-G113) and the American Cancer Society, Inc. (E-105). * Medical Student Hescarch Fellow. t Markle Scholar in Medical Science. 1 Carr, I. and Carr, .J.: Membranous whorls in the testicular interstitial cells. Anat. Rec., 144: 143-147, 19G2. 2 Christensen, A. K. and Fawcett, D. W.: The normal fine structure of opossum testicular interstitial cells . .J. Biophys. Cytol., 9: G53-G70, 19Gl. 3 Leeson, C. H.: The fine structure of rat interstitial tissue. In: Proceedings of the Fifth International Congress for Electron Microscopy. Edited by S.S. Breese, .Jr. New York: Academic Press, 1962, vol. 2. 4 Yasutaki, S.: Fine structure of the mouse testicular interstitial ceJI. In: Proceedings of the Fifth International Congress for Electron Microscopy. Edited by S.S. Breese, Jr. New York: Academic Press, 1962, vol. 2. 5 Fawcett, D. W. and Burgos, M. H.: Studies on the fine structure of the mammalian testis. IL The human interstitial tissue. Amer . .J. Anat., 107: 245-269, 1960. 6 Yamada, E.: Some observation on the fine structure of the interstitial cell in the human testis. In: Proceedings of the Fifth International Congress for Electron Microscopy. Edited by S. S. Breese, Jr. New York: Academic Press, 19G2, vol. 2. 7 Pierce, G. B., Jr.: The pathogenesis of testicular tumors. J. Urol., 88: 573-584, 1962.
Specimen contributed by R. M. Nesbit, M.D. Sniffen, R. C.: The testis. I. The normal testis. Arch. Path., 50: 259-284, 1950. *
8
G4
UJ/[R.\STR11CTURJ<1 OF H\iJ\lAi'- TESTICULAR TUJ\IOllci
(\:j
ethyl alcohols, and embedclerl in \'estopttl \\' using (.he method of Kurtz. 10 Sections 11·cn• cut on a Portp1·-Blum micmtome and stained eiiher \\it.h aqm'OlL, uranyl acetate for 24- honr., m l pl:l' cen ( lead acdatP in mctlmuol for ;-30 minute.'i. Ub'ii'n·a"liorrn 11-cre n1.ack on an RC"\. EMl--:iF del'tron micro~cope. rn:S.ULTS
1·ariatio1Js [mm extreme hypolcydigism to marked degrees of interstitial hyperplasia ,n,re anticiand encountered in thPse organs. For the ultra.st.rudural studirs, the tis,,ues were placed immediately in cold , eronal buffered osmium tetroxic!e 1Yith sucrose, 9 and minced. After l hour they were dehydrated in graded
l'lirn'it.ructurn1ly the cp]h, of tlm'iC' tumor,, \\TJ'f; similar to eacl1 ot.lwr and to nonnal inwr~lit ial cdb (fig'i. 4, 5 and (-i ,B). \Yhcrra, tlw 1rn111cl nm:lri ol' the: polygonal cclb wen· not remarkable . thP cytoplasm \Yas characterized Ow Pxti'emb development of me111hrano11.s organ('llar 8\-'it.elll'i. In many cells, ,-csicular profiles of endopla,m1ie retieulnm ,,-ere ,-:o mm1e1·ous that the cy1oplasm re.sernbled the ~urfa,cc of a tine sponge. Among the'iP ,·Psicles ,n-n· scattered mitochomlria, 1·arious osmiophilic inclnsio11,, and occasional crystalloicb of Reinke. The cells of '\1-31 (figs . .5,.-1. and G), lbe ,-irilizing tumor, 1n:re indistinguishable frnm thu.s(· of normal interstitial cells (fig. 4). The majol'ilY of eel],; of l\I-31 contained myi iads of \'(\,id<"·' ,,imila1· to those describr:cl in the interstitial cdb of llH, opossum by Christensen and Fawcett." "\.hliough t.hcsc ,·esicles had a fe11· attached ribosome.,, the,· ,,-ill be rpferred to as agranular rndopla.smie retin1lum. It is not certain whether the.,· l'epre,en\ cli,wrete n'sicle'i 01· a system of anas1omosing tubule.c; as suggested by LePson. 3 ln some C<'lls, 1d1ich contained fe,wr n•sicles and thcrcbr gan' the general impre.,sion of being less well diff('t·e11 tiated thall thr others, lamellar forms of the endoplasmic reticulum were found (fi,g. 7). Th('se profiles were: few in number and like the Ye.c,icuiar component had only a ,catt.ering of att.ar·herl ribrnmnws. Typical granular enclopla,mic 1elirnlum was rarr and eonsistcd of seat.t<·t·erl ,hmt, prnfilcs (figs. 5, B and The rnosl rlrarnaCic nwmhranous configurations were whorls 7 and 8), which were identical to those de.scribed in tJw inter,;titial cells of mice by Carr and C,1n 1 ancl by Yasutaki. 4 Ther varied in size ttrn! complexity; the simplest were composed of only une m two ronccntric, ~parscly granular, rncmbranott~ ring, (fig. 8, B), 11·hcrea, the largest wrre rnmposed of as rnany as 30 agranular rings. The~r: rings ,surrnunded a niclus, u~ually eiiJH·r a mii,0-
'Caulfield, J.B.: Effects of varying the vehicle for OsO., in tissue fixation. J. Bioph 0·s. Cytol., 3: 827-830, Hl57.
10 Kurtz, 8. AL: A nnw method for 1,mbeddinµ: tissues in \'estopal W. ,J. lJltrastruct. Hes, 5: -,m-: l(i9' HJ(i 1.
!,'10. 1. ReprnsC'ntat.ivc portion of l\f-ll, inter-..ititial eel! Lumm, illustrntes clillusc pattern of µ:rn\Tth. x:iOO.
Pru. 2 Cryst.alloicl of lfoinke surrounded ln· clear zone may be seen a1 arrow. :11-f-\J, interstiti,il cr ll wmor. x·480. 0
.Frn. 3. Organoid pattern typical of 1\l-31. X300
:1
66
BEALS, PIERCE, JR. AND SCHROEDER
Fm. 4. A, cytoplasm typical of interstitial cell from normal adult testis. Abundant branching vesicular endoplasmic reticulum forms background for mitochondria (m) and angular inclusions (a). X 18,600. B, portions of 2 interstitial cells from normal adult testis. Yesicular endoplasmic reticulum is seen as oval profiles dispersed throughout cytoplasm. n-nucleus; L-lipid droplets distorted by preparative procedures. X14,500.
T_;LTRASTRLTCTURE OF 1-rc:\IAN TESTJCULAR TL:\IORS
chonclrion or a densl: osmophilie; granule. At the of Lhe 1YJ:10rl the rnmponent membram:s appeared contimwus 1-rith the H'sicular and lamellar form~ of the endoplasmic reticulnm (fig. 8: A) In general, celb in which whorls 1Yerc obseiTed WPt·r poorly differentiated. Vifhorls wen, obc,ern-~d in lVl-31 and normal internCitial cells, but 1Yen: frequent components of the cytoplasm of ::\l-\l. Althougb prnfiles oi' the Golgi apparatL1s were seen in all ~pccimen~, tbey were oYershadowed by the Yesicular component of the cytoplasm in l\T31 and in the nornrnl interstitial Cl'lk Mito-· chondria \Ycre numerous and pkomorpl1ic, wiU1 moderately electron dense matrices that made them. appear dark in relation to their surroundings 5, 7 ancl ) . For the most pal't the cristae had the usual configuration althongh in some imtances appeared tubular (figs. 6,A. and 9,A). Of the numernus inclusiorn, obsern,cl in the~e cells, the mo~t oufatancling were Che cry.~talloiJs of Rcinke, 11 the ultrastructure of whif:h was :first described Fawcett and Bmgos. 5 They ,n:re angular in shape, not enclosed by memand their internal arrangement was that of a fine lattice. Seen in one plane, the subunits
as lon?;, thin fib rib (fig;. 9,B), while in other planes as more or less discrete symmetrically spaced, den.,c particles (fig. 9,A). i\.lthough typically only a large single crystalloid was seen in the cytoplasm, occasional cells c:ont,ained numerous smaller ones. ,\ few were observed lying free in the intracellular space. The crystalloids m formalin-fixed 11ost-osmicated t,issue were smroundecl by ntcuolated zones JO) 11-h1ch are presumably responsible for the clear space observed around crystalloids when prepared for light microscopy (fig. 2). Other inclusion~ ,aried considerably in appearance. There were ~mooth membrane-enclosed on1l or round, densely osmiophilic masses (figs. 5,B and 6,B), complex strnctnres with pleomorphic componentf (fig. 6,B), mm1crous lipid in~lusions (fig~. 4,B and 5,,1), and inclusions which were descrilied by Fawcett and Bmgos" as lipochrome pigment granules. Filamentous fascicules, .-lescribed by Fawcett and and Yamacla 6 in normal Leydig cells were not observed in the tumors. Dense, sometimes angular, inclusions often Been in tlw c~-topla.~m of cell~ containing Reinke, .F Beitrii.ge zur histologie des meuschen. Arch. f. mikr. Anut, 47: :,4--4.4, 189fi.
crystalloids of Heinke 4,--1 nnrl nw.y be precursors of c:t·ystalloids. It should be repeated that the r:elJ., nf i\L.H and the normal cells \\'NC 80 mu"h aJik(, that they could only be distiugui,,iwd thr: absence of crystaJloids in the funner the other hand, contained many whorl3, fewer profiles reticulum, and in general gave the being ll'SS ,wll-cliffercntiat.ccl than either ;\J :JJ or the normal Leydig celh DISCUSSJO.N.
electron micro,,copic st udit','-' nf I.um.ors were made in the hope 1hat consislent ulrrastructural differences might be obsen·cd betwceu neoplastic and nmmal cells that \\'ould l<'ad t,o 11 better unclerntanding of the neopla,I ic 'ik1.te. These hopes 11-ere unfu1fil1ed; in ract t-wncir, oft.en retained enough of the 1111:rastructural featun'8 of their normal counterparts to a11on establisl1rnent of histogenesis. 12 io The celb of the 2 11trnors examined in this study arc almost exan co1rnterparts of normal Leydig cells. In addition to findings 1.·eportc:d by Fa11ceU and Burgos 5 fo1· normal human inter~tit,ial whorb identical to those described Carr and 1 Carr in Lhe interstitial cells of miee lrnni bt)Pn obsern,d in the tumor~ and in normal interstitial cells. The presence of attached ribosomes (admittedly few in number) and the of the peripheral membrane,, of 1lwsc: whorls ,Yith the endoplasmic: reticulum, lead us to be· lieve that the whorl is composed of profiles uf endoplasmic reticulum ancl is, therefore, analogous to the nebenkern of tho pituitary adenornas dc:sc:rihecl b3- Oberling and Bernharcl.rn Although it is admitted that intcrpretatwn
or
1 2 Zelickson, A. S.: Electron of the basal cell epitheliorna. J.
S\,Utiy-
[)errn'.,
39: 18:3-187, HJG2. 13 Pierce, G. B., Jr. and Midgley, ,\.. H., ,Jr.· Origin and function of humm1 syncytiot rophoblastic giant cells. Amer. J. Path 43: 15~l-173,
19(i3 H Pierce, G. B., Jr., Midgley, A. ll., ,Jr., H.mn, ,). S. and Feldman, J. D.: Parietal yolk cnrcinoma: Clue 1,o the histogenesis of 'R membrane of 1.he mouse embryo. Arner. ,J. Path.
41: 549-56G, 19Ci2 . 15 Dalton, A. J., Law, L. W., J\folorn,y. J. 13. ;;.ml :VIanaker, R. A.: All electron microscopic ol a series of murine lymphoid neoplasms. J. ;,,1;. Cancer Inst., 27: 747-791, 19Gl. 16 Oberling, C. and Bernhard, W.: The morphology of the cancer cells. In: The Cell i½ed Cells, Part 2. Editerl by J. Braclrni Mirsky. Xew York: Academic Press, 19Gl, vol.+.
i
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68
F.rn. G. .i1. k,ss welJ-differnntiated of cell from 1[-31 shows portion of whorl (w) ancl mitochondria (m) with both larnellar and cristae. )(2:i,500. B, portion of neoplastic cell from !II-:ll which did not contain whorls and wbich has nrnny incltrnious and vesicles in its cytoplasm. XlS./500.
FrG. 5. A cytoplasm of neoplastic ('Pll representative of those in :\1-:31, virilizing inlerntitial cell tumor from of 7-year-olci ; Golgi bodies (g) blend with numerous profiles of vesinrbr reticulum. IVfany inclusions caa seen scattered through ; plcomorphic densf: body brnne-endosed masses (arrow J. and mitochonclrin (m). X lG,000 B, cytopla.srn of 2 adjacent cells from 11-\J interntitial cell tumor from testis of less well-diffenmtiatccl than M
70
BEALS, PIERCE, JR. AND SCHROEDER
FIG. 7. Cells illustrating degree of differentiation characteristic of M-9; majority of membranes in this figure are portions of cellular membrane. Note lack of endoplasmic reticulum and clusters of Golgi profiles. N-nucleus; w-whorl. Xl0,500.
electron micrographs can lead to erroneous functional concepts, it is worth pointing out that M-31, a virilizing tumor, contained few whorls and many vesicles of endoplasmic reticulum. Cells with whorls contained less vesicular endoplasmic reticulum than those without whorls. These data might suggest that whorls are not necessary for steroid production and may represent an inactive storage arrangement of the endoplasmic reticulum of cells which have stopped synthesizing specialized products, in this case, steroids. Porter17 has pointed out the association of steroid production and a cytoplasm containing numerous vesicular elements of agranular endoplasmic reticulum. The crystalloids of Reinke which are considered to be specific for Leydig and ovarian hilus cells have occasioned considerable speculation. The 17 Porter, K. R.: The ground substance; observations from electron microscopy. The Cell. Cells and Their Component Parts. Edited by J. Brachet and A. E. Mirsky. New York: Academic Press, 1961, vol. 2.
lattice-like structure bears a remarkable similarity to the yolk platelets in amphibian oocytes18 and the intranuclear protein crystals associated with adenovirus infections of HeLa cells. 19 Their composition and functional role in human interstitial cells have not been determined; they could represent the storage form of a specific protein or a type of abnormal secretion-product of the cells. Interstitial cells and adrenal cortical rests which are often observed in the testis have rnany features in comrnon. Crystalloids, of course, are only observed in the former. Ultrastructural studies of the adrenal cOitex under a 18 Wallace, R. A. and Karasaki, S.: Studies on amphibian yolk. 2. The isolation of yolk platelets from the eggs of Rana pipiens. J. Cell Biol., 18: 153-166, 1963. 19 .Morgan, C., Godrnan, G. C., Breitenfeld, P. M. and Rose, H. M.: A correlative study by electron and light microscopy of the development of type 5 adenovirus. I. Electron microscopy. J. Exp. Med., 112: 373-382, 1960.
Fru. 8. A and B, whorls in cytoplasm of cells from M-9 demonstrate continuity of membranes whorl with vesiculttr and lamellar components of endoplasmic reticulum. Arrow points to dense inclusion surrounded by 2 concentric membranes which could be early stage in formation of whorl.. X Hl,000 and lG,000. 71
72
BEA.LS, PIERCE, JR. A.ND SCHROEDER
Fm. 9. A, highly magnified view of crystalloid of Reinke demonstrates regularly oriented components of crystalline lattice. Arrow points to cleavage fault within crystalloid. X35,000. B, crystalloid of Reinke (er) contained within cytoplasm of interstitial cell from normal adult testis. Note large angular inclusions (a) which may be precursors of crystalloids. X17,000. variety of trophic conditions by Nishikawa and associates, 2° Carr, 21 • 22 Schwarz and asso20 Nishikawa, l\lI., l\!Iurone, I. and Sato, T.: Electron microscopic investigations of the adrenal cortex. Endocrinology, 72: 197-209, 1963. 21 Carr, I.: The ultrastructure of the human adrenal cortex before and after stimulation with ACTH. J. Path. Bact., 81: 101-106, 1961. 22 Carr, I. A.: The electron microscopy of the adrenal cortex. In: Conference on The Human Adrenal Cortex. Edited by A. R. Currie, T. Symington and J. K. Grant. Baltimore: The Williams & Wilkins Co., 1962, pp. 21-25.
ciates, 23 and Ross 24 described vesicular endoplasmic reticulum as a major component of the cytoplasm. Although it is not possible to set 23 Schwarz, W., Merker, H. J. and Suchowsky, G.: Electron microscopic studies on the effects of ACTH and stress on the adrenal cortex in rats. Virchow Arch. Path. Anat., 335: 165-179, 1962. 24 Ross, M. H.: Electron microscopy of the human foetal adrenal cortex. In: Conference on The Human Adrenal Cortex. Edited by A. R. Currie, T. Symington and J. K. Grant. Baltimore: The Williams & Wilkins Co., 1962, pp. 558-569.
ULTRASTRUCTURE OF HUMAN TESTICULAR TUMORS
73
FIG. 10. Interstitial cell from adult testis which was fixed with formalin prior to fixation for electron microscopy with osmium tetroxide. Lattice-like character of crystalloids of Reinke (er) is not evident and crystalloids are surrounded by artefactual zones of vacuolation, which could be responsible for clear spaces often seen around crystalloids prepared for light microscopy by formalin fixation. X 10,000. absolute ultrastructural criteria that distinguish adrenal cortical cells from Leydig cells, the presence of numerous microvilli and the higher frequency of lipid droplets in adrenal cortical cells and the large amount of the vesicular endoplasmic reticulum in the interstitial cells aid in the identification. Recent reports have stressed the clinical problem of differentiating a virilizing interstitial cell tumor from a hyperplastic adrenal rest in the testis of a patient with an adrenogenital syndrome.25, 26 Attempts at characterizing the testicular lesion on the basiR of steroid chemistry have not been too fruitful since an interstitial tumor that had crystalloids of Reinke and secreted testosterone contained appreciable amounts of 11-beta-hydroxylase activity, 26 an 2 5 Glenn, J. F. and Boyce, W. H.: Adrenogenitalism with testicular adrenal rests simulating interstitial cell tumor. J. Urol., 89: 456-463, 1963. 26 Savard, K., Dorfman, R. I., Baggett, B., Fielding. L. L., Engel, L. L., McPherson, H. T., Lister, L. M., Johnson, D. S., Hamblen, E. C. and Engel, F. L.: Clinical, morphological, and biochemical studies of a virilizing tumor in the testis. J. Clin. Invest., 39: 534-553, 1960.
enzyme considered to be of adrenal cortical origin. Glenn and Boyce 25 have reported that the prompt drop in level of urinary androgen;, following cortisone administration is the best evidence that a testicular nodule is a hyperplastic adrenal rest. SUMMARY
Two interstitial cell tumors are described and their ultrastructure compared with that of normal Leydig cells. The cells of the tumors and normal interstitial tissue are characterized by a cytoplasm containing numerous \ esicular elements of endoplasmic reticulum, and a variety of inclusions. One of the tumors showed numerous crystalloids of Reinke. In the other crystalloids were not seen, but the cells were otherwise indistinguishable from those of normal Leydig cells. The authors acknowledge help and criticism of Reed M. Nesbit, M.D., and B. Naylor, M.D., as well as technical assistance of Miss Faith Pruchnicki, Miss Jessica Goodwin and Mr. Matthew Harden.