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6J mice
Experimental Gerontology, Vol. 19, pp. 313-320, 1984
0531-5565/84 $3.00 + .00 Copyright©1984Pergamon Press Ltd
Printed in the USA.All rights reserved.
THE DEVELOPMENT OF MAMMOTROPH ADENOMAS IN PITUITARIES OF AGING FEMALE C57BL/6J MICE
CLAUDIA J. CLAYTON*, JOELSCHECHTER** and CALEBE. FINCH~ *Department of Neurology University of Rochester School of Medicine Rochester, New York **Department of Anatomy University of Southern California School of Medicine Los Angeles, California and ~tDepartment of Biological Sciences and The Andrus Gerontology Center University of Southern California Los Angeles, California
(Received 7 December 1983) Abstract-The development of pituitary adenomas in female C57BL/6J mice was studied over the
lifespan. Mammotrophs and somatotrophs, identified by immunocytochemical staining, appeared normal in 3-4 month old animals, with the exception of an occasional hypertrophic mammotroph and, in one gland, a nest of such cells. At 15 months, hypertrophic mammotrophs, somatotrophs and unstained cells occurred singly or in areas of hyperplasia; a mammotrophic adenoma was present in one gland. At 22 months, pituitaries contained mammotroph adenomas or adenomalike mammotroph clusters within hypertrophic, hyperplastic areas as seen in 15 month old glands. Pituitaries from all 28-30 month old mice contained mammotroph adenomas; nests of hypertrophic cells were present in 5 of 8 glands studied. These results suggest that mammotrophic adenomas may arise from hypertrophic mammotrophs, which are often found in nests with other hypertrophic pituitary cell types. However, this study does not exclude the possibility that mammotroph adenomas and hypertrophic mammotrophs occur independently. INTRODUCTION SPOr~TANEOUS ADENOMAS o f the a n t e r i o r p i t u i t a r y c o m m o n l y o c c u r d u r i n g r e p r o d u c t i v e senescence in m a n y l a b o r a t o r y r o d e n t s ( D u c h e n an d Sch u r r , 1976). In f e m a l e C 5 7 B L / 6 J m i c e (Felicio et aL, 1980), p i t u i t a r y a d e n o m a s are rare b e f o r e 15 m o n t h s , b u t are f o u n d in m o s t mice by 24 m o n t h s . S u c h t u m o r s h a v e been c h a r a c t e r i z e d by c o n v e n t i o n a l histological staining m e t h o d s , e l e c t r o n m i c r o s c o p y , a n d m o r e recently, i m m u n o c y t o c h e m i c a l t e c h n i q u e s (Ito e t al., 1972b; K o v a c s et al., 1977; S c h e c h t e r et al., 1981; T i b o l d i et al., 1967; also, see K o v a c s et al., 1977b f o r a review). T h e m a j o r i t y o f cells in the t u m o r s appear to be m a m m o t r o p h s a n d s o r n a t o t r o p h s , a l t h o u g h s o m e a u t o n o m o u s t u m o r strains 313
314
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contain cells which produce both prolactin (Prl) and growth hormone (GH) (Furth et al., 1973; Ito et al., 1972a; Ueda et al., 1973). In a recent report, we identified the mammotroph in the pituitary tumors of old C57BL/6J mice by structural characteristics (Schechter et al., 1981). Grossly elevated plasma prolactin (Prl), to > 500 ng/ml) is c o m m o n in old rats with pituitary tumors (Huang et al., 1976). In contrast, plasma Prl of old C57BL/6J mice with gross tumors tends to remain low ( < 100 ng/ml) and is elevated to > 150 ng/ml only in a subgroup of those mice with large tumors (Nelson et al., 1980). However, in view of the absence of the general hyperprolactinemia of old tumor-bearing mice and the degranulated appearance of many mice pituitary cells (Schechter et al., 1981), immunocytochemical characterization with specific antisera was required to establish tumor cell type as mammotrophic. The structural and immunocytochemical characteristics of mammotrophs were evaluated throughout the 30-month average lifespan of C57BL/6J mice. By 15 months, ovarian cyclicity is lost and is succeeded by a polyfollicular anovulatory condition during which plasma estradiol is sustained at modest levels while progesterone is very low (Nelson et al., 1981). During the polyfollicular phase, the incidence of grossly observable tumors increases many-fold, from < 10°70 at 14 months to > 40°7o by 24 months (Felicio et al., 1980). After 24 months, plasma estradiol drops to castrate levels in association with ovarian depletion (Gosden et al., 1983), whereas plasma L H becomes elevated to castrate levels (Gee et al., 1983). The hypertrophied gonadotrophs seen in this age group (Schechter et al., 1981) are consistent with the elevated L H seen in other samplings (Gee et al., 1983). Between 24 and 30 months, the incidence of tumors frequently is > 60°7o (Felico et al., 1980). Importantly, L H was not elevated if pituitary tumors were present (Gee et al., 1983). Thus, C57BL/6J mice display a striking model of menopause in humans involving similar decreases in plasma estradiol and elevations of LH. Because mammotrophic tumors can be induced by chronic estrogen treatment in young rats (Clifton and Meyer, 1956; Sonnenschein et al., 1974) and in young mice (Giok 1961), the steady but modest plasma estradiol experienced during the polyfollicular status just after the loss of cyclicity by 15 months could be a factor in tumorigenesis. It was therefore of interest to characterize m a m m o t r o p h morphology at these major phases of reproductive senescence, to acquire further background for the construction of hypotheses on the origins of mammotrophic tumors. Additionally, we were able to evaluate if Prl and GH occurred in the same pituitary tumor cells, because both Prl and GH were reported in some autonomous tumor lines ( F u r t h e t al., 1973); Ito et al., 1972a; Ueda et al., 1973), and because of evidence that 3-week treatments with estradiol can transform somatotrophs into m a m m o t r o p h s in young rats (Stratmann et al., 1974). MATERIALS AND METHODS Pituitaries from four age groups of female C57BL/6J mice were studied: 3-4 months (n = 7), 15 months (n = 7), 22 months (n = 6) and 28-30 months (n = 8). Mice were obtained from the Jackson Laboratory (Bar Harbor, ME) as virgins (3 months) or retired breeders (8 months) and maintained as previously described (Holinka and Finch, 1977). Glands were obtained by two methods: 1) After decapitation, the pituitaries were exposed and flooded with Bouin's solution or 4% neutral buffered paraformaldehyde in situ; or, 2) mice were anesthetized with sodium pentobarbital and perfused through the left cardiac ventricle with saline followed by 4% paraformaldehyde. Pituitaries were then removed, fixed, dehydrated by alcohol, embedded in paraffin, sectioned at 6urn on a rotary mierotome, and mounted on gelatin coated slides. Immunocytochemicalstaining and absorption controls were performed using mouse Prl, mouse GH, rabbit anti-mouse Prl and monkeyanti-mouse GH (Sinha et al., 1972). Tissue sections were stained using the unlabelled antibody-enzymemethods (Sternberger 1970). Sections were incubated for 24 hours with anti-Prl or anti-GH at dilutions of 1:1400and 1:1000, respec-
315
M A M M O T R O P H A D E N O M A S IN C 5 7 B I / 6 J MICE T A B L E 1. T H E PRESENCE OF ABNORMAL CELLS IN THE PITUITARIES OF FEMALE
C57BL/6J
MICE AS A F U N C T I O N OF AGE.
Age
# o f Glands with Scattered Single Hypertrophic Mammotrophs
# o f Glands with Areas o f Hypertrophy and Hyperplasia (multiple cell types)
# o f Glands with Mammotroph Adenomas
n
3-4 m o n t h s 15 m o n t h s 22 m o n t h s 28-30 m o n t h s
4 2 0 4
1 5 5 5
0 1 6 8
7 7 6 8
tively. Adjacent sections were incubated with anti-Prl that was preabsorbed with Prl or GH anti-GH that was preabsorbed w i ~ Prl, or anti-rabbit g a m m a globin ( A A R G O 1:50, and peroxidase-anti-peroxidase (PAP), 1:200. Finally, sections were exposed to 3,3'-diaminobenzidine, 30 rag%, in 0.01 M Tris buffer pH 7.4, containing 0.03% H~O2, then dehydrated and m o u n t e d with coverslips.
RESULTS Abnormal cell types as a function of age (described below) are summarized in Table 1. 3-month old mice
Most mammotrophs and somatotrophs at 3 months had normal morphology. The mammotrophs were irregular in shape and demonstrated the pockets of stained cytoplasm characteristic of this cell type (Figure 1). The somatotrophs were round or ovoid, with
L
FIG. 1. Normal 3-month-old pituitary stained for Prl, showing pockets of stained cytoplasm typical of m a m motrophs (arrows). 6 t* section. ( x 450). FI6. 2. Section near to that in Figure 1, stained with anti-Prl that was pre-absorbed with Prl. Although some background staining was present, no specific cytoplasmic staining was observed. 6 # section. ( x 450).
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c . J . CLAYTON, J. S C H E C H T E R , AND C.E. FINCH
c y t o p l a s m i c staining which was m o r e u n i f o r m l y d i s t r i b u t e d a b o u t the nuclei t h a n that o f the m a m m o t r o p h s . Single h y p e r t r o p h i c m a m m o t r o p h s a n d s o m a t o t r o p h s were seen occasionally at this age, a n d o n e p i t u i t a r y c o n t a i n e d a nest o f h y p e r t r o p h i c m a m m o t r o p h s similar to t h o s e seen at later ages. P r e a b s o r p t i o n o f the p r i m a r y a n t i s e r a with their respective antigens e l i m i n a t e d specific c y t o p l a s m i c staining, a l t h o u g h s o m e b a c k g r o u n d staining was present w h e n a n t i - P r l was p r e a b s o r b e d with Prl ( F i g u r e 2). C r o s s - a b s o r p t i o n s , (e.g., a n t i - P r l p r e a b s o r b e d with G H a n d a n t i - G H p r e a b s o r b e d with Prl) gave the s a m e results as for sections s t a i n e d with u n t r e a t e d p r i m a r y antisera. W h e n sections stained for Prl were c o m p a r e d with a d j a c e n t sections s t a i n e d f o r G H , n o evidence was seen o f a n y cells cont a i n i n g b o t h h o r m o n e s . T h e a b o v e results e x t e n d to the o t h e r age g r o u p s a n d will not be r e p e a t e d below.
15-month old mice Single h y p e r t r o p h i c m a m m o t r o p h s a n d s o m a t o t r o p h s o c c a s i o n a l l y were p r e s e n t a m o n g n o r m a l - a p p e a r i n g cells, b u t the o u t s t a n d i n g f e a t u r e o f the p i t u i t a r i e s at 15 m o n t h s was the
FIG. 3. 15-month old pituitary stained with anti-GH, showing a hyperplastic lesion containing hypertrophic somatotrophs (small arrows), somatotrophs in an abnormal configuration (large arrow), and many unstained cells. Some normal appearing somatotrophs are present at the periphery of the lesion (medium arrows). 6 u section. (x 450). FIG. 4. Section near to that in Figure 3, showing hyperplastic lesion stained for Prl. Hyperplastic mammotrophs are present (arrows; compare with normal lactotrophs in Figure 1 at the same magnification) with numerous unstained cells. Some disruption and enlargement of vascular sinuses is apparent, with colloidal material remaining in some of the spaces (asterisks). This section was counterstained with methylene blue because the immunocytochemical background staining was insufficient for observation of unstained cells. 6 u section. ( x 450).
MAMMOTROPH
A D E N O M A S IN C 5 7 B L / 6 J MICE
317
presence o f a r e a s o f h y p e r p l a s i a in m o s t ( 5 / 7 ) g l a n d s . These h y p e r p l a s t i c a r e a s c o n t a i n e d m a m m o t r o p h s a n d s o m a t o t r o p h s t h a t u s u a l l y were h y p e r t r o p h i c or in a b n o r m a l conf i g u r a t i o n s ( F i g u r e s 3 a n d 4), as well as large n u m b e r s o f u n s t a i n e d cells. O n e p i t u i t a r y also c o n t a i n e d a small a d e n o m a r e s e m b l i n g the large t u m o r s seen in the o l d e r age g r o u p s , as d e s c r i b e d below. V a s c u l a r lakes were present, u s u a l l y in the f o r m o f thin slits p e n e t r a t i n g b e t w e e n rows o f t u m o r cells; s o m e larger lakes were o b s e r v e d .
22-month old mice P i t u i t a r i e s at 22 m o n t h s h a d c h a r a c t e r i s t i c a r e a s o f h y p e r t r o p h y a n d h y p e r p l a s i a cont a i n i n g m a m m o t r o p h s , s o m a t o t r o p h s a n d u n s t a i n e d cells as seen at 15 m o n t h s . H o w e v e r , these h y p e r p l a s t i c a r e a s o f t e n c o n t a i n e d nests o f m a m m o t r o p h s which, a l t h o u g h n o t clearly h y p e r t r o p h i c , d i f f e r e d f r o m n o r m a l m a m m o t r o p h s in s h a p e a n d d i s t r i b u t i o n o f s t a i n e d c y t o p l a s m ( F i g u r e 5). T h e s e cells r e s e m b l e d in size a n d s h a p e those f o u n d in f r a n k a d e n o m a s ( F i g u r e 6) in this a n d o t h e r age g r o u p s . T h e a d e n o m a s c o n t a i n e d c o r d s a n d clusters o f cells, n e a r l y all o f which s t a i n e d lightly or d a r k l y with a n t i - P r l . S o m a t o t r o p h s were r a r e l y f o u n d within these a d e n o m a s , V a s c u l a r lakes were c o m m o n ( F i g u r e 5) a n d sometimes contained hypertrophic or adenomatous mammotrophs.
Fic. 5. Hyperplasia in a 22-month-old pituitary, stained for Prl. Hypertrophic mammotrophs are present (arrows) and nests of smaller mammotrophs (boxes) are similar in size and appearance to cells of mammotroph adenomas (see Figure 6). Asterisks denote vascular lakes filled with colloidal material. FI~. 6. Adenomatous region from the same pituitary as in Figure 5, stained for Prl. The cells are arranged in cords and clusters typical of an adenoma, and virtually all are stained. The morphology of this tumor is similar to that of adenomas seen in other age groups. Small vascular lakes are indicated by asterisks, 6 v section. ( × 450).
318
c . J . ('1 A Y T O N , J. S C H E C H T E R , A N D C.E. F I N C H
dt
%
'~'°
if
Fro. 7. Low power illustration of a mammotroph adenoma in a 28-month-oldpituitary. The tumor is fairly well circumscribed, and some invasion of the neural lobe (N) has occurred. The tumor is riddled with small vascular lakes (arrows), which are poorly resolved at this low power. 6 ~ section. (× 93). Fro. 8. Photomontage of a section adjacent to Figure 7, stained for GH. Few somatotrophs are present within the body of the tumor (arrows), although they are numerous at its periphery. The neural lobe is demarcated by N. 6 # section ( × 93).
2 8 - 3 0 - m o n t h old mice M a m m o t r o p h adenomas similar to those in the 22-month age group were present in 100% of the oldest mice. These tumors often invaded the neural lobe (Figure 7); several expanded beyond the sella turcica, to indent the hypothalamus as observed during removal of the pituitary from the skull. Such tumors were encapsulated and were easily separated from adjacent neural tissue. Again, somatotrophs within these tumors were rare (Figure 8). Tumors were riddled with vascular lakes, ranging from slit-like spaces to large, colloid-filled areas loosely lined with Prl-containing cells. By gross observation of these and younger animals, there were no secondary tumor loci in the CNS or the periphery. DISCUSSION The pituitary adenomas which arise spontaneously during aging in female C57BL/6J mice consist mainly o f mammotrophs, as established by immunocytochemical procedures with antibodies to mouse Prl. These results thus establish the previous hormonal assignment of these tumors in C57BL/6J mice, which was based on ultrastructural and tinctorial properties (Schechter et aL, 1981). No evidence suggested that G H and Prl occurred concomitantly within the same cell; absorption controls indicated that crossreactive staining did not occur between the two hormones. However, some studies have demonstrated pituitary adenoma cells which produce more than one hormone (Furth et al., 1973; Ito et al., 1972; Ueda et al., 1973). Interestingly, multihormonal cells usually were observed in induced or transplanted tumors, rather than in spontaneous tumors or normal pituitaries; however, Ueda et al. (1973) reported the existence of rare multihormonal cells in the latter. Although artifacts from crossreactivity of the antisera might be a factor in the findings of other investigators, it remains possible that pituitary tumor cells could produce both hormones.
MAMMOTROPH ADENOMASIN C57BL/6J MICE
319
Numerous unstained cells were present in the hypertrophic, hyperplastic lesions. All or some of these cells may be gonadotrophic, somatotrophic or mammotrophic; the absence of immunocytochemical staining was perhaps due to previous hormone release or to hormone synthesis in quantities too small to allow immunocytochemical observation (Schechter et al., 1981). In any case, the occurrence of hypertrophy and hyperplasia of somatotrophs and mammotrophs, as well as hypertrophy of gonadotrophs (Schechter, unpublished), appear to precede the development of frank mammotroph adenomas. These data, considered with observations of adenoma-like clusters of mammotrophs within areas of hypertrophy and hyperplasia, suggest that mammotroph adenomas may arise from hypertrophic mammotrophs. Although no somatotrophic adenomas were observed here, some GH-containing tumors were found in another immunocytochemical study of aged mouse pituitaries (Schechter, Felicio, Nelson and Finch, unpublished). No gross gonadotrophic tumors were detected in C57BL/6J mice. It is intriguing that the adenomas contained few somatotrophs and no other hyperplastic cell types were observed (such cells would have been negatively stained in this study). Thus, the stimuli causing hyperplasia of several cell types may be distinct from those causing subsequent adenoma development. It remains possible that mammotroph adenomas arise independently of hypertrophic mammotrophs. No secondary tumors were detected by gross observation. The presence of vascular lakes within tumor zones is an indication of capillary disruption (Schechter et al., 1981). Although some tumors in the older animals extended beyond the sella between the brain and skull, these clearly were encapsulated with no evidence for metastasis. In general, spontaneous pituitary tumors are not metastatic (Berry and Caplan, 1979). Acknowledgements-We thank Dr. Y.N. Sinha for his generous gifts of mouse Prl, anti-mouse Prl, mouse GH and anti-mouse GH, Dr. Ludwig Sternberger for his gift of PAP, and Dr. Lowell Lapham for valuable advice. The authors also thank Joanne M. Bell for technical assistance and Mary Croucher and Tamara Badger for assistance in the preparation of the manuscript. This work was supported by N1A Young Investigator Award to C.J.C (AG-01903), by NIA grants to C.E.F. (AG-00117; AG-00447), by grants to J.E.S. from the NCI (CA-21426) and by HEW-NIH grant CA-14089 to Los Angeles County/USC Cancer Center.
REFERENCES BERRY, R.G. and CAPLAN, H.J. (1979) Ann. Clin. Lab. Sci. 9: 94-102. CLIFTON, K.H. and MEYER, R.K. (1956) Anat. Rec. 125: 65-81. DUCHEN, L.W. and SCHURR, P.H. (1976) In, Hypothalamus, Pituitary and Aging. (A.V. Everitt and J.A. Burgess, eds.) Thomas, Springfield, Ill., pp. 137-156. FELICIO, L.S., NELSON, J.N. and FINCH, C.E. (1980) Exp. Geront. 15: 139-142. FURTH, J., UEDA, G. and CLIFTON, K.H. (1973) In, Methods in Cancer Research. (H. Busch, ed.) Academic Press, New York, Vol. 10, Ch 8, pp. 201-277. GEE, D.M., FLURKEY, K. and FINCH, C.E. (1983) Biol. Repro. 28: 598-607. GIOK, K.H. (1961) A n Experimental Study of Pituitary tumors: Genesis, Cytology and Hormone content. Springer-Verlag, Berlin. GOSDEN, R.G., LIANG, S.C., FELIClO, L.S., NELSON, J.F. and FINCH, C.E. (1983) Biol. Repro. 28: 225-260. HOLINKA, C.F. and FINCH, C.E. (1977) BioL Repro. 16: 385-393. HUANG, H.H., MARSHALL, S. and MEITES, J. (1976) Biol. Repro. 14: 538-543. ITo, A., FURTH, J. and MoY, P. (1972a) Cancer Res., 32: 48-56. Iro, A., MOY, P., KAUmTZ, K., KORTWRIOHT, K., CLARKE, S., FURTH, J. and MEITES, J. (1972b) J. Natl. Cancer Inst., 49: 702-711. KOVACS, K., HORVATH, E. and EZRIN, C. (1977a) Path Anm., 12, Part 2: 341-382. KOVACS, K., HORVATH, E., ILSE, R.G., IZRIN, C. and ILSE, D. (1977b) Beitr. Path., 161: 1-16.
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NELSON, J.F., FELICIO, L.S., OSTI~RBURG,H.H., and FINCH, C.E. (1981) Biol. Repro. 24: 784-794. NELSON, J.F., FELICIO, L.S., SINHA, Y.N., and FINCH, C.E. (1980) The Gerontologist 20:171 (abstract). SCHECHTER, J., FELICIO, L., NELSON, J.F. and FINCH, C.E. (1981) Anat. Rec., 199: 423-432. SINHA, Y.N., LEWIS, U.J. and VANDERLAAN, W.P. (1972) J. Endocr., 55: 31-40. SONNENSCHEIN,C., POSNER,i . , SAHR,K., FAROOKHI, R., and BRUNELLE, R. (1974)Exp. CelI Res. 84:339-411 STERNBERGER, L.A. (1970) J. Histochem. Cutochem. 18: 315-333. STRATMANN, I.E., EZYIN, C., and SELLERS, E.A. (1974) Cell Tiss, Res. 152: 229-238. TIBOLDI, T., VIRAGH, S., KovAcs, K., HODI, M. and JULESZ, M. (1967) J. Microscopic Sci., 6: 677-689. UEDA, G., MOY, P. and FURTH, J. (1973) Int. J. Cancer, 12: 100-114.
0531-5565/84 $3.00 + .00 Copyright©1984Pergamon Press Ltd
Printed in the USA.All rights reserved.
THE DEVELOPMENT OF MAMMOTROPH ADENOMAS IN PITUITARIES OF AGING FEMALE C57BL/6J MICE
CLAUDIA J. CLAYTON*, JOELSCHECHTER** and CALEBE. FINCH~ *Department of Neurology University of Rochester School of Medicine Rochester, New York **Department of Anatomy University of Southern California School of Medicine Los Angeles, California and ~tDepartment of Biological Sciences and The Andrus Gerontology Center University of Southern California Los Angeles, California
(Received 7 December 1983) Abstract-The development of pituitary adenomas in female C57BL/6J mice was studied over the
lifespan. Mammotrophs and somatotrophs, identified by immunocytochemical staining, appeared normal in 3-4 month old animals, with the exception of an occasional hypertrophic mammotroph and, in one gland, a nest of such cells. At 15 months, hypertrophic mammotrophs, somatotrophs and unstained cells occurred singly or in areas of hyperplasia; a mammotrophic adenoma was present in one gland. At 22 months, pituitaries contained mammotroph adenomas or adenomalike mammotroph clusters within hypertrophic, hyperplastic areas as seen in 15 month old glands. Pituitaries from all 28-30 month old mice contained mammotroph adenomas; nests of hypertrophic cells were present in 5 of 8 glands studied. These results suggest that mammotrophic adenomas may arise from hypertrophic mammotrophs, which are often found in nests with other hypertrophic pituitary cell types. However, this study does not exclude the possibility that mammotroph adenomas and hypertrophic mammotrophs occur independently. INTRODUCTION SPOr~TANEOUS ADENOMAS o f the a n t e r i o r p i t u i t a r y c o m m o n l y o c c u r d u r i n g r e p r o d u c t i v e senescence in m a n y l a b o r a t o r y r o d e n t s ( D u c h e n an d Sch u r r , 1976). In f e m a l e C 5 7 B L / 6 J m i c e (Felicio et aL, 1980), p i t u i t a r y a d e n o m a s are rare b e f o r e 15 m o n t h s , b u t are f o u n d in m o s t mice by 24 m o n t h s . S u c h t u m o r s h a v e been c h a r a c t e r i z e d by c o n v e n t i o n a l histological staining m e t h o d s , e l e c t r o n m i c r o s c o p y , a n d m o r e recently, i m m u n o c y t o c h e m i c a l t e c h n i q u e s (Ito e t al., 1972b; K o v a c s et al., 1977; S c h e c h t e r et al., 1981; T i b o l d i et al., 1967; also, see K o v a c s et al., 1977b f o r a review). T h e m a j o r i t y o f cells in the t u m o r s appear to be m a m m o t r o p h s a n d s o r n a t o t r o p h s , a l t h o u g h s o m e a u t o n o m o u s t u m o r strains 313
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contain cells which produce both prolactin (Prl) and growth hormone (GH) (Furth et al., 1973; Ito et al., 1972a; Ueda et al., 1973). In a recent report, we identified the mammotroph in the pituitary tumors of old C57BL/6J mice by structural characteristics (Schechter et al., 1981). Grossly elevated plasma prolactin (Prl), to > 500 ng/ml) is c o m m o n in old rats with pituitary tumors (Huang et al., 1976). In contrast, plasma Prl of old C57BL/6J mice with gross tumors tends to remain low ( < 100 ng/ml) and is elevated to > 150 ng/ml only in a subgroup of those mice with large tumors (Nelson et al., 1980). However, in view of the absence of the general hyperprolactinemia of old tumor-bearing mice and the degranulated appearance of many mice pituitary cells (Schechter et al., 1981), immunocytochemical characterization with specific antisera was required to establish tumor cell type as mammotrophic. The structural and immunocytochemical characteristics of mammotrophs were evaluated throughout the 30-month average lifespan of C57BL/6J mice. By 15 months, ovarian cyclicity is lost and is succeeded by a polyfollicular anovulatory condition during which plasma estradiol is sustained at modest levels while progesterone is very low (Nelson et al., 1981). During the polyfollicular phase, the incidence of grossly observable tumors increases many-fold, from < 10°70 at 14 months to > 40°7o by 24 months (Felicio et al., 1980). After 24 months, plasma estradiol drops to castrate levels in association with ovarian depletion (Gosden et al., 1983), whereas plasma L H becomes elevated to castrate levels (Gee et al., 1983). The hypertrophied gonadotrophs seen in this age group (Schechter et al., 1981) are consistent with the elevated L H seen in other samplings (Gee et al., 1983). Between 24 and 30 months, the incidence of tumors frequently is > 60°7o (Felico et al., 1980). Importantly, L H was not elevated if pituitary tumors were present (Gee et al., 1983). Thus, C57BL/6J mice display a striking model of menopause in humans involving similar decreases in plasma estradiol and elevations of LH. Because mammotrophic tumors can be induced by chronic estrogen treatment in young rats (Clifton and Meyer, 1956; Sonnenschein et al., 1974) and in young mice (Giok 1961), the steady but modest plasma estradiol experienced during the polyfollicular status just after the loss of cyclicity by 15 months could be a factor in tumorigenesis. It was therefore of interest to characterize m a m m o t r o p h morphology at these major phases of reproductive senescence, to acquire further background for the construction of hypotheses on the origins of mammotrophic tumors. Additionally, we were able to evaluate if Prl and GH occurred in the same pituitary tumor cells, because both Prl and GH were reported in some autonomous tumor lines ( F u r t h e t al., 1973); Ito et al., 1972a; Ueda et al., 1973), and because of evidence that 3-week treatments with estradiol can transform somatotrophs into m a m m o t r o p h s in young rats (Stratmann et al., 1974). MATERIALS AND METHODS Pituitaries from four age groups of female C57BL/6J mice were studied: 3-4 months (n = 7), 15 months (n = 7), 22 months (n = 6) and 28-30 months (n = 8). Mice were obtained from the Jackson Laboratory (Bar Harbor, ME) as virgins (3 months) or retired breeders (8 months) and maintained as previously described (Holinka and Finch, 1977). Glands were obtained by two methods: 1) After decapitation, the pituitaries were exposed and flooded with Bouin's solution or 4% neutral buffered paraformaldehyde in situ; or, 2) mice were anesthetized with sodium pentobarbital and perfused through the left cardiac ventricle with saline followed by 4% paraformaldehyde. Pituitaries were then removed, fixed, dehydrated by alcohol, embedded in paraffin, sectioned at 6urn on a rotary mierotome, and mounted on gelatin coated slides. Immunocytochemicalstaining and absorption controls were performed using mouse Prl, mouse GH, rabbit anti-mouse Prl and monkeyanti-mouse GH (Sinha et al., 1972). Tissue sections were stained using the unlabelled antibody-enzymemethods (Sternberger 1970). Sections were incubated for 24 hours with anti-Prl or anti-GH at dilutions of 1:1400and 1:1000, respec-
315
M A M M O T R O P H A D E N O M A S IN C 5 7 B I / 6 J MICE T A B L E 1. T H E PRESENCE OF ABNORMAL CELLS IN THE PITUITARIES OF FEMALE
C57BL/6J
MICE AS A F U N C T I O N OF AGE.
Age
# o f Glands with Scattered Single Hypertrophic Mammotrophs
# o f Glands with Areas o f Hypertrophy and Hyperplasia (multiple cell types)
# o f Glands with Mammotroph Adenomas
n
3-4 m o n t h s 15 m o n t h s 22 m o n t h s 28-30 m o n t h s
4 2 0 4
1 5 5 5
0 1 6 8
7 7 6 8
tively. Adjacent sections were incubated with anti-Prl that was preabsorbed with Prl or GH anti-GH that was preabsorbed w i ~ Prl, or anti-rabbit g a m m a globin ( A A R G O 1:50, and peroxidase-anti-peroxidase (PAP), 1:200. Finally, sections were exposed to 3,3'-diaminobenzidine, 30 rag%, in 0.01 M Tris buffer pH 7.4, containing 0.03% H~O2, then dehydrated and m o u n t e d with coverslips.
RESULTS Abnormal cell types as a function of age (described below) are summarized in Table 1. 3-month old mice
Most mammotrophs and somatotrophs at 3 months had normal morphology. The mammotrophs were irregular in shape and demonstrated the pockets of stained cytoplasm characteristic of this cell type (Figure 1). The somatotrophs were round or ovoid, with
L
FIG. 1. Normal 3-month-old pituitary stained for Prl, showing pockets of stained cytoplasm typical of m a m motrophs (arrows). 6 t* section. ( x 450). FI6. 2. Section near to that in Figure 1, stained with anti-Prl that was pre-absorbed with Prl. Although some background staining was present, no specific cytoplasmic staining was observed. 6 # section. ( x 450).
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c y t o p l a s m i c staining which was m o r e u n i f o r m l y d i s t r i b u t e d a b o u t the nuclei t h a n that o f the m a m m o t r o p h s . Single h y p e r t r o p h i c m a m m o t r o p h s a n d s o m a t o t r o p h s were seen occasionally at this age, a n d o n e p i t u i t a r y c o n t a i n e d a nest o f h y p e r t r o p h i c m a m m o t r o p h s similar to t h o s e seen at later ages. P r e a b s o r p t i o n o f the p r i m a r y a n t i s e r a with their respective antigens e l i m i n a t e d specific c y t o p l a s m i c staining, a l t h o u g h s o m e b a c k g r o u n d staining was present w h e n a n t i - P r l was p r e a b s o r b e d with Prl ( F i g u r e 2). C r o s s - a b s o r p t i o n s , (e.g., a n t i - P r l p r e a b s o r b e d with G H a n d a n t i - G H p r e a b s o r b e d with Prl) gave the s a m e results as for sections s t a i n e d with u n t r e a t e d p r i m a r y antisera. W h e n sections stained for Prl were c o m p a r e d with a d j a c e n t sections s t a i n e d f o r G H , n o evidence was seen o f a n y cells cont a i n i n g b o t h h o r m o n e s . T h e a b o v e results e x t e n d to the o t h e r age g r o u p s a n d will not be r e p e a t e d below.
15-month old mice Single h y p e r t r o p h i c m a m m o t r o p h s a n d s o m a t o t r o p h s o c c a s i o n a l l y were p r e s e n t a m o n g n o r m a l - a p p e a r i n g cells, b u t the o u t s t a n d i n g f e a t u r e o f the p i t u i t a r i e s at 15 m o n t h s was the
FIG. 3. 15-month old pituitary stained with anti-GH, showing a hyperplastic lesion containing hypertrophic somatotrophs (small arrows), somatotrophs in an abnormal configuration (large arrow), and many unstained cells. Some normal appearing somatotrophs are present at the periphery of the lesion (medium arrows). 6 u section. (x 450). FIG. 4. Section near to that in Figure 3, showing hyperplastic lesion stained for Prl. Hyperplastic mammotrophs are present (arrows; compare with normal lactotrophs in Figure 1 at the same magnification) with numerous unstained cells. Some disruption and enlargement of vascular sinuses is apparent, with colloidal material remaining in some of the spaces (asterisks). This section was counterstained with methylene blue because the immunocytochemical background staining was insufficient for observation of unstained cells. 6 u section. ( x 450).
MAMMOTROPH
A D E N O M A S IN C 5 7 B L / 6 J MICE
317
presence o f a r e a s o f h y p e r p l a s i a in m o s t ( 5 / 7 ) g l a n d s . These h y p e r p l a s t i c a r e a s c o n t a i n e d m a m m o t r o p h s a n d s o m a t o t r o p h s t h a t u s u a l l y were h y p e r t r o p h i c or in a b n o r m a l conf i g u r a t i o n s ( F i g u r e s 3 a n d 4), as well as large n u m b e r s o f u n s t a i n e d cells. O n e p i t u i t a r y also c o n t a i n e d a small a d e n o m a r e s e m b l i n g the large t u m o r s seen in the o l d e r age g r o u p s , as d e s c r i b e d below. V a s c u l a r lakes were present, u s u a l l y in the f o r m o f thin slits p e n e t r a t i n g b e t w e e n rows o f t u m o r cells; s o m e larger lakes were o b s e r v e d .
22-month old mice P i t u i t a r i e s at 22 m o n t h s h a d c h a r a c t e r i s t i c a r e a s o f h y p e r t r o p h y a n d h y p e r p l a s i a cont a i n i n g m a m m o t r o p h s , s o m a t o t r o p h s a n d u n s t a i n e d cells as seen at 15 m o n t h s . H o w e v e r , these h y p e r p l a s t i c a r e a s o f t e n c o n t a i n e d nests o f m a m m o t r o p h s which, a l t h o u g h n o t clearly h y p e r t r o p h i c , d i f f e r e d f r o m n o r m a l m a m m o t r o p h s in s h a p e a n d d i s t r i b u t i o n o f s t a i n e d c y t o p l a s m ( F i g u r e 5). T h e s e cells r e s e m b l e d in size a n d s h a p e those f o u n d in f r a n k a d e n o m a s ( F i g u r e 6) in this a n d o t h e r age g r o u p s . T h e a d e n o m a s c o n t a i n e d c o r d s a n d clusters o f cells, n e a r l y all o f which s t a i n e d lightly or d a r k l y with a n t i - P r l . S o m a t o t r o p h s were r a r e l y f o u n d within these a d e n o m a s , V a s c u l a r lakes were c o m m o n ( F i g u r e 5) a n d sometimes contained hypertrophic or adenomatous mammotrophs.
Fic. 5. Hyperplasia in a 22-month-old pituitary, stained for Prl. Hypertrophic mammotrophs are present (arrows) and nests of smaller mammotrophs (boxes) are similar in size and appearance to cells of mammotroph adenomas (see Figure 6). Asterisks denote vascular lakes filled with colloidal material. FI~. 6. Adenomatous region from the same pituitary as in Figure 5, stained for Prl. The cells are arranged in cords and clusters typical of an adenoma, and virtually all are stained. The morphology of this tumor is similar to that of adenomas seen in other age groups. Small vascular lakes are indicated by asterisks, 6 v section. ( × 450).
318
c . J . ('1 A Y T O N , J. S C H E C H T E R , A N D C.E. F I N C H
dt
%
'~'°
if
Fro. 7. Low power illustration of a mammotroph adenoma in a 28-month-oldpituitary. The tumor is fairly well circumscribed, and some invasion of the neural lobe (N) has occurred. The tumor is riddled with small vascular lakes (arrows), which are poorly resolved at this low power. 6 ~ section. (× 93). Fro. 8. Photomontage of a section adjacent to Figure 7, stained for GH. Few somatotrophs are present within the body of the tumor (arrows), although they are numerous at its periphery. The neural lobe is demarcated by N. 6 # section ( × 93).
2 8 - 3 0 - m o n t h old mice M a m m o t r o p h adenomas similar to those in the 22-month age group were present in 100% of the oldest mice. These tumors often invaded the neural lobe (Figure 7); several expanded beyond the sella turcica, to indent the hypothalamus as observed during removal of the pituitary from the skull. Such tumors were encapsulated and were easily separated from adjacent neural tissue. Again, somatotrophs within these tumors were rare (Figure 8). Tumors were riddled with vascular lakes, ranging from slit-like spaces to large, colloid-filled areas loosely lined with Prl-containing cells. By gross observation of these and younger animals, there were no secondary tumor loci in the CNS or the periphery. DISCUSSION The pituitary adenomas which arise spontaneously during aging in female C57BL/6J mice consist mainly o f mammotrophs, as established by immunocytochemical procedures with antibodies to mouse Prl. These results thus establish the previous hormonal assignment of these tumors in C57BL/6J mice, which was based on ultrastructural and tinctorial properties (Schechter et aL, 1981). No evidence suggested that G H and Prl occurred concomitantly within the same cell; absorption controls indicated that crossreactive staining did not occur between the two hormones. However, some studies have demonstrated pituitary adenoma cells which produce more than one hormone (Furth et al., 1973; Ito et al., 1972; Ueda et al., 1973). Interestingly, multihormonal cells usually were observed in induced or transplanted tumors, rather than in spontaneous tumors or normal pituitaries; however, Ueda et al. (1973) reported the existence of rare multihormonal cells in the latter. Although artifacts from crossreactivity of the antisera might be a factor in the findings of other investigators, it remains possible that pituitary tumor cells could produce both hormones.
MAMMOTROPH ADENOMASIN C57BL/6J MICE
319
Numerous unstained cells were present in the hypertrophic, hyperplastic lesions. All or some of these cells may be gonadotrophic, somatotrophic or mammotrophic; the absence of immunocytochemical staining was perhaps due to previous hormone release or to hormone synthesis in quantities too small to allow immunocytochemical observation (Schechter et al., 1981). In any case, the occurrence of hypertrophy and hyperplasia of somatotrophs and mammotrophs, as well as hypertrophy of gonadotrophs (Schechter, unpublished), appear to precede the development of frank mammotroph adenomas. These data, considered with observations of adenoma-like clusters of mammotrophs within areas of hypertrophy and hyperplasia, suggest that mammotroph adenomas may arise from hypertrophic mammotrophs. Although no somatotrophic adenomas were observed here, some GH-containing tumors were found in another immunocytochemical study of aged mouse pituitaries (Schechter, Felicio, Nelson and Finch, unpublished). No gross gonadotrophic tumors were detected in C57BL/6J mice. It is intriguing that the adenomas contained few somatotrophs and no other hyperplastic cell types were observed (such cells would have been negatively stained in this study). Thus, the stimuli causing hyperplasia of several cell types may be distinct from those causing subsequent adenoma development. It remains possible that mammotroph adenomas arise independently of hypertrophic mammotrophs. No secondary tumors were detected by gross observation. The presence of vascular lakes within tumor zones is an indication of capillary disruption (Schechter et al., 1981). Although some tumors in the older animals extended beyond the sella between the brain and skull, these clearly were encapsulated with no evidence for metastasis. In general, spontaneous pituitary tumors are not metastatic (Berry and Caplan, 1979). Acknowledgements-We thank Dr. Y.N. Sinha for his generous gifts of mouse Prl, anti-mouse Prl, mouse GH and anti-mouse GH, Dr. Ludwig Sternberger for his gift of PAP, and Dr. Lowell Lapham for valuable advice. The authors also thank Joanne M. Bell for technical assistance and Mary Croucher and Tamara Badger for assistance in the preparation of the manuscript. This work was supported by N1A Young Investigator Award to C.J.C (AG-01903), by NIA grants to C.E.F. (AG-00117; AG-00447), by grants to J.E.S. from the NCI (CA-21426) and by HEW-NIH grant CA-14089 to Los Angeles County/USC Cancer Center.
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