[CANCER RESEARCH 36, 3748-3760, October 1976]

SUMMARY

Bilateral orchidectomy performed on 10- to 15-day-oldWistan mats,immediately followed by the homogmaft of onetestis into the splenopancreatic epiploon, resulted 6 monthslater in leydigiomas at the site of the graft in 100% of theanimals. After this latency period, the tumors continued togrow proportionately to time elapsed and were associatedwith both pituitary microadenomas of the aldehyde-fuchsin-, aldehyde-thionin-pemiodic acid-Schiff-, and periodicacid-Schiff-positive cells and diffuse hyperplasia of thepamathyroids, affecting in particular the chief cells of thelatter.

After a 6- on 12-month leydigioma latency period, the ratswere given a daily treatment by gavagefor 45daysof one ontwo of the following steroids: Compound A, 17a-ethynyl,(5a)-androst-2-en, 17f3-ol (17f3-acetate) (5 mg/kg whenalone; 2.5 mg/kg when combined); Compound B, 17a-ethynyl, (5a)-androst-2-en, 17f3-oI (17f3-nicotinate) (always givenin combined form at 2 mg/kg); Compound C, 16-oximinoestrone, 3-allyl ether (2 mg/kg when combined). The treatments caused, in various degrees, (a) involution of theleydigiomas until complete disappearance in up to 50% ofthe cases; (b) recovery of the characteristic morphologicalpattern of the pituitanies; and (c) cytolysis and interstitialfibrosis in the previously hypenplastic parathynoids.

INTRODUCTION

Spontaneous testicular tumors can exist in certain strainsof laboratory animals (10); however, experimentally inducedtumors have obvious advantages in endocrine oncology(12, 18, 33). These tumors are most frequently induced bythe administration of various products such as estrogens (8,11, 15, 21), tniphenylethylene (9), or methylcholanthrene(22). Other methods relate either to experimental cryptomchidy (3) or to auto- (29) or heterografts (4). In particular, theleydigiomas were obtained practically exclusively bychronic endogenous stimulation of the gonadotmophic pituitary (2). As reported in the papers of Milcu et a!. (20) andPetnea and Zimel (29), bilateral orchidectomy performed onrats less than 15 days old, immediately followed by the

I Present address: Clinique Endocrinologique, Faculté de Mödecine Pitié

Salpetrière, HOpital de Ia Pitié,75013 Paris, France.Received February 17, 1976; accepted July 7, 1976.

autograft of only 1 testis into the splenopancreatic epiploon, causes the development of a testicular tumor withinterstitial cells within 6 months. Twelve months after thegraft, compression of the abdominal organs with ascites isobserved. Oeath can also be caused by the concomitantdevelopment of gonadotrophic extensive and hemorrhagicpituitary adenomas.

In order to determine the activity of some specific steroids(Table 1) on the evolution of leydigiomas, we observed notonly the existence of gonadotrophic pituitary hyperplasiasbut also the development of extensive pamathynoidhyperplasias that regressed under the effect of the treatment, at thesame time as the involution of the leydigiomas and thepituitary hyperplasias. Intriguing but possible, this reactioncould be related to the proliferative polyglandulam syndromes, the biological and clinical peculiarities of whichare recently starting to be better understood (17).

MATERIALS AND METHODS

Male Wistar rats were castrated between the ages of 10and 15 days, and 1 of the 2 testes was immediately graftedinto the splenopancreatic epiploon.

The development of the graft was controlled by explonatory laparotomy and biopsies before the onset of the treatment, either 6 months later (rats of Groups 3 and 4), or 12months later (rats of Groups 5 to 8; see Table 1).

The animals thus prepared, as well as the uncastratedcontrols of the same age, were grouped and treated daily bygavage for 45 days (Table 1). The specific androgens andestrogen compounds (Lasdon Foundation, ColoradoSprings, Cob. , and ThéramexS.A. , Paris, France), as previously studied, were utilized because of their gonad-inhibiting activities (13, 30, 34, 35).

On the 46th day after treatment, all the animals weresacrificed, including the controls of the same age (8 and 14months, respectively). The leydigiomas were removed andweighed. Fragments of leydigiomas, pituitanies, and thynopanathymoidcomplex were removed and prepared for histological and cytological study with light and electron microscopy (fixation in 3.5% glutaraldehyde buffered with Venonalphosphate at pH 7.4, postfixed in 1% 0s04 and thenembedded in Epon. The blocks were sliced with a leadcitrate-umanyl acetate sequence and examined with a B11Hitachi electron microscope at an accelerating voltage of 50to 80 kV).

3748 CANCERRESEARCHVOL. 36

Hyperplasia of the Parathyroids and the Pituitary in Ratswith Experimental Leydigiomas and Inhibitory Effectof Several Steroids

I. Petrea and N. Guéritée1Department of internal Medicine, Griffin Hospital, Derby, Connecticut, 06418

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Ratswenetreated daily by gavagefor 45 days.Ageof ani

malsatendof ex

Experimentalconditions andNo.ofperimentGrouptreatmentappliedrats(mos.)1Intact

controls1582lntactcontnols15143Leydigiomas

after 6 months;nontreatedcontrols1084As

in Group 3; treatment withCompounds A@+C1585Leydigiomas

after 12 months;nontreatedcontrols3146As

in Group 5; treatment withCompound8147As

in Group 5; treatment withCompounds A +C8148Asin Group 5; treatment with

Compounds B + C814

7-

Ratio of the number of testicular graft lyses and thanimals/group

Rats were treated daily by gavage for 45 days.e

numberofNo.

of aniNo. of tes

ticularGroupmals Treatmentgraftlyses5

3Control068 CompoundA478 Compounds A +C388 Compounds B + C4

Parathyroid and Pituitary Hyperplasia in Leydigiomas

Table 1

Animal groups, experimentalconditions, and treatment applied<0.001) in comparison with nontreated controls. The meanweight of the leydigiomas of the controls sacrificed 14months after the graft (Table 1, Group 5) was 2.533 ±0.260g (Chart 1). The histological examination of the tumorshowed nameseminiferous tubules with no epithelial contentand advanced tubular fibrosis. The tumor consisted practically entirely of large cells with either dense on vacuolancytoplasm and ovoid or round nuclei resembling luteal-hikecells (Fig. 2). In electron microscopy (Figs. 3, 4), 2 categonies of Leydig cells are apparent. The first are larger withdense cytoplasm, a dense ergastoplasmic system, numerous mitochondnia, but a few secretory granules. The others,on the contrary, are clear cells and smaller, probably comesponding to aging or due to functional depletion factors.Here the ergastoplasm is scanty, the vacuohization is sometimes observed, and the mitochondnias are far less numerous and often seem degenerated. Inhibition of the leydigiomas after 45 days of treatment with Compounds A, A +C, and B + C (Table 1, Groups 6 to 8) often caused disappearance of the grafts, as can be seen in Table 2.

Thus, the mean weight of the tumors of the treated animals was reduced in a highly significant manner (Chart 1;Table 2). The inhibition of the leydigiomas, which had notcompletely disappeared under the influence of the treatment when examined histologically, consisted of the presence of degenerative phenomena, karyopyknosis, and hyalinofibrosis (Figs. 5, 6, and 9). The fluorescence of theleydigioma cells stained with acnidine orange is slight in the

_3.000k‘@ I

U) 2.000

Cl)

@ 1.000

@ 0.100@

t- 0.080

@ 0.0600

C, 0.040

@;0.020 H@1

u9.87 u@9.84 u@9.60p <0.001 <0.001 <0.001

a Compound A: 17a-ethynyl, (5a)-androsten-2-en, 17/3-01 (17/3-

acetate) (5 mg/kg when alone, or 2.5 mg/kg when combined);Compound B: 17a-ethynyl, (5a)-andnost-2-en , 17/3-oh (17f3-nicotinate); (given only in combined form at 2 mg/kg); Compound C: 16-oximinoestrone, 3 allyl ether (given only in combined form at 2 mg/kg).

Some pituitary sections were stained with fuchsin-aldehyde (23) and thionin-PAS2 aldehyde (5).

Fragments of leydigiomas were also prepared for fluorescent microscopy according to the method of Bertalanffi andNagy, as modified in Ref. 28. Sections were stained with0.10% acnidine orange, diluted to 1/10,000 in a phosphatesolution buffered at pH 6, and then incubated in a 0.1 Msolution of CaCI2. They were examined with a UV fluonescent lamp (400-watt HBO and UG 1/35 filter).

Because the parathynoids are embedded in the thyroidpanenchyma, we have done serial histological sections ofthe whole thyroid-parathyroid complex, and we selectedonly the slides on which the panathyroids were the largest indiameter. The slides were projected with a Leitz projectorwith an identical objective lens and focal distance. Thus itwas possible to trace the contours of the thyroid and parathyroids and to compare their differences planimetrically(25).

RESULTS

Leydigiomas. All the controls sacrificed 8 months afterthe graft of the testis (Table 1, Group 3) developed tumorswith a mean weight of 0.887 ±0.248 g. There was nospenmatogenesis; the seminiferous tubules were atrophicand the interstitial cells showed active proliferation (Fig. 1).After 45 days of treatment with Compounds A + C (Table 1,Group 4), the testicular tumors were inhibited up to totaldisappearance of the grafts in 8 of 15 cases. Hence, themean weight of the tumors was reduced (0.034 ±0.0049 g)in the treated group in a highly significant manner (p

2 The abbreviation used is: PAS, periodic acid- Schiff.

Chart 1. Leydigioma tumor mass (g) of male Wistar rats 14 months old atthe end of the experiment, leydigiomas;@, leydigiomas and treatmentwith 17a-ethynyl (5a)-androst-2-en , 17/3-01(1713-acetate);Ifflflll, leydigiomasand treatment with 17a-ethynyl, (5a)-androst-2-en, 17@-ol(17p-acetate) + 16-oximinoestrone, 3-allyl ether;E@j@',leydigiomas and treatment with l7cx-ethynyl, (5a)-androst-2-en , 17/3-01(17f3-nicotinate) + 16-oximinoestrone, 3-allylether.

Table 2

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I. Petrea and N. Guéritée

grafts with a latency period of 8 months (Table 1, Group 3;Fig. 7). However, it was intensive in the leydigioma cellsafter a latency period of 14 months (Table 1, Group 5; Fig.8). Under the influence of the treatments, when fibrosis wasextensive, the fluorescence was practically nonexistent(Fig. 10), marking possibly an inhibition of the RNA synthesis (28).

Pituitary. The histological study of the animals of Groups3 and 5 (Fig . 11; Table 1) revealed the presence of certainlarge cells that are sometimes completely vacuolam, sometimes containing PAS-,aldehyde-fuchsin-,and thion in-aIdehyde-positive granules. The cellular distribution is diffuse orsometimes nodular.

In electron microscopy (Figs. 12 to 15), the secretorygranules sometimes exceed 200 nm and are usually situatednear the cellular membrane. The number of granules isinversely proportionate to the volume and active prohifemation of the leydigioma. The ergastoplasmic system of thepituitary cells is progressively inhibited. The meticular membranes can break, thus transforming the cytoplasm into amultivacuolam structure which gives a “clearcell' ‘appearance.

The pituitanies ofthe treated animals (Figs. 16 to 18) showvarious degreesof remission, up to the complete disappearance of the “signetring-like cells―which, in the nontreatedcontrols, showed different degrees of cellular hypertmophyand secretory activity. Electron microscopy reveals theabundance of secretory granules which seem relatively uniform and remain within taxonomic limits characteristic ofgranules of the gonadotrophic type, with their 2-dimensional variables, SG1 < 200 nm; SG2 > 200 nm (Figs. 17 and18).

Parathyroids. In leydigioma-beaning animals, the panathyroids are obviously enlarged and expand in the thyroidtissue. The treatment with the applied steroids, whateverthe type, causes a decrease in volume of the pamathynoidsand of their proliferation into the thyroid gland (Fig. 19).

The histological examination (Table 1, Group 5) of theleydigioma-beaning controls shows diffuse hyperplasia ofthe panathyroid (Figs. 20 and 21) with numerous chief-likecells having dense, basophihic cytoplasm, alternating withlange, light, slightly eosinophihic cells (Fig. 21). In thetreated animals (Figs. 22 to 24, 28, and 29), the variousdegrees of inhibition of the parathynoid hypemplasiaareobserved. The inhibition is characterized by nuclear andcytoplasmic degenerative phenomena leading to interstitialfibrosis.

In electron microscopy (Figs. 25 to 27), the presence ofvariable cells, which are probably forms of a functionaltransition, was observed in the parathyroids of the leydigioma-beaning controls. Some of the chief cells containsecretory granules (Fig. 25) that measure about 200 nm.However, other “dark―-Iikecells with practically no secretory granules contain a dense endoplasmic reticulum, numemousnibosomesand polysomes, an important system ofmicrovesicles with a sometimes osmiophihic content, andactive, round, or oval mitochondrias (Figs. 26 and 27). The“light―cells, on the contrary, more frequently show degenerative and involutional signs such as reduction of the engastoplasmic system and nibosomes and complete absence

of the secretory granules (Fig. 27). The most characteristicaspects of the cellular involution of the parathyroids underthe influence of the treatment are the bizarrely shapednuclei and the presence of numerous lysosomes (Figs. 30and 31).

DISCUSSION

We have just shown how the induction in matsof a Leydigcell tumor, after early castration and immediate autograft of1 testis in the portal vein area, causes the development of

hyperplasias in 2 other glands, the pituitary and panathynoids, and how these other hyperplasias regress at the sametime as the leydigioma under the effect of the appropriatetreatment.

The development of pituitary microadenomas with large,light cells containing dilated endoplasmic reticulum withprogressive loss of secretory granules could be consideredin the context of chronic pituitary stimulation triggered offby castration (2, 14, 18, 19, 26-29, 33). One of the histological expressions of this stimulation is the appearance of“signetring cells―(6)which correspond to a pituitary cellulamexhaustion due to their holocnine-Iike function.

The new phenomenon observed consists of a constantparathymoid hypemplasia which evolves at the same time asdoes the testicular tumor (and the pituitary hyperplasia) andwhich might even be dependent. Otherwise, it would bedifficult to understand the active cytolysis and interstitialfibrosis observed in the panathyroids of animals in whichleydigiomas regressed on disappeared under the effect ofthe various applied treatments.

One of the questions to be raised could regard the biologyof the hyperplastic parathynoid.A pamathynoid-hikehormonesecreted by a spontaneous transplanted Leydig cell tumorhas recentlybeen reportedinrats.Thishormone causeslethal hypercalcemia and hypercalciumia and , in thymopamathynoidectomized animals, the increase of the phosphorusclearance. This substance, which does not pass the panebiotic barmier, is neither analogous to the panathonmone,nor to the 0 vitamins, nor to an osteolytic phytosterol.Furthermore, it does not seem that the disturbances in thecalcium metabolism of these matsare associated with anatomic modifications of the pamathyroids(31). We, therefore,find no analogy between this later observation and ours,which, however, does not exclude the intervention of theparathommoneon calcemia (16), non that, possibly, of theleydigioma (37). A paraneoplasia-hike syndrome (17, 24)could be linked with the development of the leydigioma andrevealed by both diffuse hypemplasiaof the panathyroidsandhypercalcemia. However, another unknown mechanismcannot be ruled out.

A gonadotmophicstimulation as intense as the one causedby very early castration, and continually progressive, couldend in a modified hormone-secreting phenotype (36). Suchcells would be likely to secrete intermediate hormone substanceswhich, deviatedfrom the normal sequenceof physiological end hormones, could stimulate others than thespecific ones, the pamathyroidsfor instance (32).

Whatever the case, careful verification of the intermediate

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Parathyroid and Pituitary Hyperp!asia in Leydigiomas

hormonal substances' nature or of the ectopic hormones isnecessary.Confirming this hypothesisare the observationsof the Leeds team (1, 7) which report the frequency ofosteolytic hypemparathymoidism in postmenopausal womenor after ovamiectomy,comparable to those found in leydigioma-beaningmatsafter chronic and progressiverunawayactivity of the gonadotmophicpituitary.

As for the action of the 3 steroid compounds (CompoundsA, B, and C) (Table 1), used alone or in combined forms, thelatter had the most potent inhibitory effect against leydigiomas, micronodular gonadotnophic pituitanies, andassociated hyperplasia of the pamathyroidglands. It wouldappear that the steroids are mainly acting at the pituitarylevel, on which leydigiomas are dependent. However, at thistime we have no proof that the atrophy of leydigiomas alonecould headto the involution of the hypemplastic pamathynoidsor how extensive the moleplayed by the steroids in theseexperimental conditions could be.

REFERENCES

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14. Hopkins, C. R., and Farquhar IA. G. Hormone Secretion by Cells Dissociated from Rat Anterior Pituitaries. J. Cell Biol., 59: 276-303, 1973.

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Fig. 1. Leydigioma 8 months after bilateral orchidectomy and testicular graft, showing complete inhibition of spermatogenesis, seminiferous tubules (Si)with degenerate Sertoli cells (DS), and proliferation of the interstitial cells (IC). Trichromic stain, x 350.

Fig. 2. Leydigioma 14 months after bilateral orchidectomy and testicular graft. The microscopic field is completely occupied by clear interstitial cells (CIC),as well as large cells with dark cytoplasm. Some mitotic figures (MF) could be observed. Trichromic stain, x 350.

Figs. 3 and 4. Same as in Fig. 2. The ultrastructure of leydigioma cells with dense cytoplasm (DIC) and numerous mitochondria (M). Other cells are clearinterstitial cells (CIC) with free ribosomes (FR) or lacunar endoplasmic reticulum (V). N, nucleus; CM, cell membrane. ER, endoplasmic reticulum. x 24,000and x 12,000.

Fig. 5. Leydigioma 8 months after bilateral orchidectomy and testicular graft. The animal was treated 45 days with the combination of Compounds A + C(Table 1, Group 4). An atrophic seminiferous tubule (ST) surrounded by numerous pyknotic interstitial cells (IC) with vacuobization and hyalinosis of thecytoplasm. Trichromic stain, x 350.

Fig. 6. Leydigioma 14 months after bilateral orchidectomy and testicular graft. The animal was treated for 45 days with Compounds A + C (Table 1, Group7). There is obvious fibrosis (IF) progressing into the remnant of degenerated interstitial cells (IC). Tnchromic stain, x 350.

Fig. 7. The same as in Fig. 1. Pale fluorescence of the interstitial cells (IC). ST, seminiferous tubule. UV, x 300.Fig. 8. Same case as in Fig. 2. Intensive fluorescence of interstitial cell cytoplasm (IC). UV, x 300.Fig. 9. Leydigioma 14 months after bilateral orchidectomy and testicular graft. The animal was treated for 45 days with Compounds B + C (Table 1, Group

8). There is intensive fibrosis (IF) of the tumor with almost complete degeneration and replacement of the interstitial cells (IC) by connective tissue. STseminiferous tubule. Trichromic stain, x 350.

Fig. 10. The same as in Fig. 9. The fluorescence in the interstitial fibrosis (IF) and degenerated remnant interstitial cells is minimal. UV, x 300.Fig. 11. Pituitary of the case presented in Fig. 2. There are numerous gonadotrophic cells (GC) with signet ring-like appearance. Trichromic stain, X 300.Figs. 12 to 15. Pituitary gonadotrophic cells of different rats bearing leydigioma tumor 14 months after the testicular graft and bilateral orchidectomy. The

cells have a clear cytoplasm with lacunar endoplasmic reticulum (LEA) and scanty secretory granules (SG, SG1, and SG@,),generally located at the cellularperiphery. Mitochondrias (M) are round, some degenerated; ER, endoplasmic reticulum; L, lysosome; NM, nuclear membrane; NP, nuclear pore; N, nucleus.x 8,000 to 24,000.

Fig. 16. Pituitary of the case presented in Fig. 9. There is a restitution ofthe pituitary gland parenchyma with absence of the signet ring-like cells, 45 days oftreatment with Compounds B + C (Table 1, Group 8). Trichromic stain, x 300.

Figs. 17 and 18. Gonadotrophic cells in leydigioma-bearing animals after 45 days of treatment with Compounds A + C (Fig. 17) and B + C (Fig. 18). Thegranular content of the cytoplasm is increased and both SGi and SGa secretory granules are abundantly secreted as well as enlarged mitochondrias (M). N,nucleus; NP, nuclear pore; ER, endoplasmic reticulum; and FR, free ribosomes. x 24,000.

Fig. 19. Thyroparathyroidian complex. Representative serial sections involving the largest circumference of the parathyroid glands in: I, controls; Ii,leydigiomas; lii, leydigiomas; treatment with 17a-ethynyl, (5a)-androst-2-en, 17f3-ob(17$-acetate) + 16-oximincestrone, 3-ablyl ether. Leitz microprojector,objective, x 15 to 26 from 15-cm distance.

Fig. 20. Parathyroid gland hyperplasia (P) infiltrating the thyroidian parenchyma (TF) in an animal bearing beydigioma 14 months after bilateral orchidectomy and graft of the testis (Table 1, Group 5). The parathyroidian hyperplastic cells are proliferating in solid sheets and consist of elements with densecytoplasma. Trichromic stain, x 500.

Fig. 21. Parathyroidian gland hyperplasia in an animal (same experimental group as in Fig. 20) where a diffuse proliferation with clear(CPC) and dark chieflike cells (PCC) are seen. Semifine section after Epon embedding. Toluidine blue, x 500.

Fig. 22. Hyperplastic parathyroid gland in a rat with leydigioma treated with the Compound A (Table 1, Group 6). The parathyroid cells (P) are pyknotlc anddislocated by connective trabeculae (IF). Trichromic stain, x 275.

Fig. 23. Hyperplastic parathyroid gland in a rat with leydigioma treated with combination of Compounds A + C (Table 1, Group 7). The remnantdegenerated parathyroidian cells (P) are replaced or surrounded by extensive fibrosis (IF). TF, thyroidian follicle. Trichromic stain, x 275.

Fig. 24. Hyperplastic parathyroidian gland in a rat with leydigioma treated with a combination of Compounds B + C (Table 1, Group 8). The parathyroidiancells (P) are pyknotic and replaced by intensive fibrosis (IF). Trichromic stain, x 375.

Fig. 25. Chief cell-like parathyroidian tissue with secretory granules (SG) in a rat with leydigioma; N, nucleus. x 24,000.Fig. 26. Hyperplastic parathyroidian cell in a rat with beydigioma. The cytoplasm has a well-developed ergastoplasm (ER), numerous mitochondrias (M),

and several microvesicles (MV). CM, cellular membrane. x 24,000.Fig. 27. Rat with leydigioma. Clear (CPC) and dark cells (PCC) without secretory granules in a hyperplastic parathyroid. The dark cell contains a dense

matrix with numerous ribosomes (A), mitochondria (M), and polyribosomes (PR), as well as microvesicbes (MV), some filled with a dense osmiophilic material.FR, free ribosomes (FR); ER, endoplasmic reticulum; CM, cellular membrane. x 24,000.

Figs. 28 and 29. Involution of hyperplastic parathyroids in rats with leydigiomas after 45 days of treatment with Compounds A + C (Fig. 28) and B + C (Fig.29). P, parathyroidian cells; IF, interstitial fibrosis. Trichromic stain, x 350.

Figs. 30 and 31. Same cases as in Figs. 28 and 29. Bizarre forms of nuclei (N) and numerous lysosomes (L) with degenerate mitochondrias (M)characterize the cellular ultrastructure of the hyperplastic parathyroidian cells in leydigiomas after treatment. DC. dense cytoplasm; V microvesicles. x18,000.

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1976;36:3748-3760. Cancer Res   I. Petrea and N. Guéritée  SteroidsExperimental Leydigiomas and Inhibitory Effect of Several Hyperplasia of the Parathyroids and the Pituitary in Rats with

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