JOURNAL OF PATHOLOGY, VOL.

165: 11 1-1 18 (1991)

THE EXPRESSION OF PARATHYROID HORMONE MESSENGER RNA IN NORMAL AND ABNORMAL PARATHYROID TISSUE c. H. KENDALL,

PHILLIPA A. ROBERTS*, J. H. PRINGLE* AND I. LAUDER*

Department of Histopathology, Leicester Royal Injrmary, Leicester LEI 5 WW, U.K.; *University ofleicester, Department of Pathology, Clinical Sciences Building, Leicester Royal Injrmary, P.O. Box 65, Leicester LE2 7LX, U.K. Received5 December 1990 Accepted 18 April 1991

SUMMARY The distribution and expression of preproparathyroid hormone (PTH) mRNA were investigated in parathyroid tissue from 57 parathyroidectomy specimens. PTH mRNA was detected by in situ hybridization using digoxigeninlabelled oligonucleotide probes. Cell morphology was seen to correlate with PTH mRNA expression. Strong expression of PTH mRNA was confined to cells which on haematoxylin and eosin staining had large vesicular nuclei. These included both vacuolated and non-vacuolatedcells. Chief cells with small dark nuclei and scanty cytoplasm had little or no expression. In both adenoma and chief cell hyperplasia, the striking difference from normal was the greatly increased proportion of cells expressing PTH mRNA. In adenomas, the rim of uninvolved parathyroid tissue showed PTH mRNA expression similar to that of normal parathyroid. In hyperplasia, there was frequently concordance of staining within individual nodules. The findings establish morphological criteria for activity of parathyroid tissue and support current concepts of the different pathogenesis of hyperplasia and adenoma. The expression of PTH mRNA in oxyphil change and parathyroid carcinomas was also investigated. KEY

WORDS-parathyroid, messenger RNA, in situ hybridization.

INTRODUCTION In the normal and abnormal parathyroid gland, chief cell function as it relates to morphology has long remained difficult to study. This is largely a result of the lack of a suitable marker of chief cell activity. Methods for the detection of stored hormone have received most attention,’.’ but there are technical difficulties. Histochemical investigations have also not proved fully e f f e ~ t i v e .Most ~ ’ ~ ofwhat is currently known has been obtained from comparative light and electron microscopy s t ~ d i e s Obvious .~’~ limitations, however, inherent in the microsampling involved have hampered more detailed analysis. The development of in situ hybridization techniques has enabled the localization of specific Addressee for correspondence: Dr C. H. Kendall, Department of Histopathology, Leicester Royal Infirmary, Leicester LEI 5WW, U.K.

0022-3417/91/1001114)8 $05.00 0 1991 by John Wiley & Sons, Ltd.

mRNAs in tissue sections. In this study, preproparathyroid hormone (PTH) mRNA was detected in routine paraffin sections using a cocktail of digoxigenin-labelled synthetic oligonucleotide probes.’ The hybrids were then visualized for light microscopy immunocytochemically with a polyclonal anti-digoxigenin alkaline phosphatase conjugate. We have investigated the localization of PTH mRNA in parathyroid chief cells. The aim was to assess the potential value of the techniques as a measure of cellular synthetic function. Diagnostically in parathyroid pathology, a number of questions remain. These include the controversy of adenoma versus hyperplasia, the nature and synthetic activity of oxyphil cells, and the criteria for parathyroid malignancy. We have therefore also investigated a range of abnormal parathyroid tissue to assess the contribution of this technique in addressing these questions.

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MATERIALS AND METHODS Preparation of tissues and sections Fresh surgical specimens were immersed for 24-48 h in 10 per cent formol saline prior to routine paraffin wax processing. This consisted of tissue from 54 parathyroidectomy specimens and included 7 normal, 25 hyperplasia, 18 adenomas, and 4 carcinomas. Normal parathyroid tissue was mainly derived from that incidentally removed in thyroidectomy specimens. Microscope slides were cleaned using the method described by Pringle et a1.8 and paraffin-embedded tissue sections were cut at 4-5 pm onto slides coated with 3-aminopropyltriethoxysilane and left overnight to dry at 37°C. These sections were dewaxed and rehydrated through a series of clean xylene, ethanol, and diethylpyrocarbonate-treated ultrapure water (DEPC-water). All solutions and equipment used for the pretreatments and hybridization stages were appropriately treated to remove nucleases, especially RNase which is ubiquitous and heat-stable. Pretreatments Pretreatments are required to unmask mRNA target sequences and to permeabilize the tissue sections for the detection rea ents. These included those performed by Cox et al. and were carried out as previously described." Briefly, the slides were immersed into 2 x SSC buffer (SSC is 150 mM NaC1, 15 mM trisodium citrate, pH 7.0) at 70°C for 10 min and transferred to 50mM Tris-HC1, pH 7.65, in DEPC-water (Tris-HC1). The sections were then digested in proteinase K (Boehringer Mannheim, F.R.G., 161519) solutions in Tris-HCI at concentrations ranging from 0.5 to 10pg/ml and incubated at 37°C for 1 h. Following digestion, the sections were washed in DEPC-water at 4°C for 10 min ( x 2). When required, RNase pretreatment controls were carried out at this stage. RNase type 1A (DNase-free) (Sigma, U.K., R4875) digests were performed at 100 pg/ml in 2 x SSC/I 0 mM MgCl, at 37°C in coplin jars for 1 h. Slides not receiving all the pretreatments were incubated in 2 x SSC/lO mM MgClJDEPC for the same time and temperature. Finally, the sections were post-fixed in 0.4 per cent paraformaldehyde, 0. I M phosphate-buffered saline/ DEPC at 4°C in precooled solutions and washed in DEPC-water ( x 2).

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0ligon ucleo t ide syn thesis Synthetic oligonucleotides were synthesized by phosphoramidite chemistry'' using a DNA synthesizer model 380B (Applied Biosystems, U.S.A.) based on the cDNA sequence for preproparathyroid hormone as described by Hendy et a1.'* Eight oligonucleotides 30 bases long were prepared on 0.2 pmol columns from sequence antisense to preproparathyroid hormone mRNA. Base additions were made using 0-(2 - cyanoethy1)-N,N-diisopropylphosphoramidites (Cruachem, U.K.) as described by McBride et al.I3 Oligonucleotide labelling Oligonucleotides were labelled at the 3' end using the TdT reaction previously described by Pringle et aL8 to add a homopolymer tail of dUTP-11digoxigenin. This reaction is based on the improved procedure for homopolymer tailing using TdT as described by Deng and W U . ' ~ Hybridization andpost-hybridization washing The hybridization and post-hybridization conditions were as previously described." The sections were drained, excess fluid was removed and the slides were then covered in 25 p1 of prehybridization buffer containing 50 per cent (v/v) formamide, 600 mM NaCl. The sections were prehybridized for 1 h at 37°C in a humid chamber in a hot-air incubator. After prehybridization the slides were drained and the sections were covered with 50 pl of prewarmed hybridization buffer containing labelled probe at 0.2 ,ug/ml. The sections were then covered with siliconized coverslips (dimethyldichlorosilane) in order to ensure even coverage with the hybridization solution. The slides were then returned to the humid chambers and incubated at 37°C overnight. Following hybridization, the coverslips were removed from the sections by transferring the slides to 2 x SSCj50 per cent (v/v) formamide prewarmed to 37°C. The sections were then washed with the following post-hybridization schedule: 2 x SSC/50 per cent (v/v) formamide at 37°C in a water bath (3 x 10 min) ; 2 x SSC at room temperature (4 x 1 min). Finally, the sections were washed in Trisbuffered saline (TBS) [50 mM Tris-HCl,0.15 M NaC1, 2 mM MgCl, 0.1 per cent (w/v) bovine serum albumin (Sigma, U.K., A7906), pH 7.61 containing 0.1 per cent (v/v) Triton X-100 at room temperature for 15 min.

PARATHORMONE mRNA EXPRESSION IN PARATHYROID TISSUE

Detection A direct detection method using anti-digoxigenin alkaline phosphatase conjugate (Boehringer Mannheim, F.R.G.) (1 :600) was employed. The sections were incubated in detection reagents, diluted in TBS, in a moist chamber at room temperature for 30 min. Between incubations, the sections were washed in TBS ( x 2). Following the immunocytochemical procedures, alkaline phosphatase activity was demonstrated using the 5-bromo-4-chloro-3indolyl phosphate (BCIP enzyme substrate) and nitroblue tetrazolium (NBT chromogen) method as previously described.* After incubation in the demonstration solution, the sections were washed in ultra-pure water, counterstained lightly in haematoxylin, and mounted in Apathy’s medium.

Specijicity Specificity of PTH mRNA staining in tissue sections was established by positivity in parathyroid tissue being confined to chief cells. A number of other glandular tissues including thyroid were entirely negative. The oligonucleotide cocktails

Fig. I-In situ hybridization detection of parathormone mRNA in parathyroid tissue. An inactive area (left) compared with an active area (right)

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were also evaluated for the detection of specific mRNA by Northern dot blotting. Total RNA was extracted from normal spleen and parathyroid tissue following the procedure of Chirgwin et a1.,I5 as modified by Freeman et a1.I6 and Northern blotting was carried out using nitrocellulose filters. Oli onucleotide cocktail probes were labelled with [a- PIdCTP using TdT, which adds a radioactive nucleotide to the 3’-hydroxyls. The hybridization and post-hybridization conditions were the same as those for the slides.

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RESULTS

PTH mRNA expression in relation to cell morphology Throughout the diRerent tissues examined, there was a consistent pattern of PTH mRNA expression in relation to cell morphology. Chief cells with small dark nuclei and scanty cytoplasm on haematoxylin and eosin staining uniformly had little or no mRNA expression. Strong cytoplasmic mRNA expression was seen only in cells with larger vesicular nuclei (Figs 1 and 2.). These included both non-

Fig. 2-Serial section of Fig. 1 stained with H & E. The cells in the inactive area have small, darkly stained nuclei and scanty cytoplasm compared with those in the active area which have larger, more vesicular nuclei and abundant cytoplasm

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vacuolated cells and cells with vacuolation (Fig. 3). Even cells which were almost totally vacuolated often showed positivity adjacent to the plasma membrane. Oxyphil cells had greatly reduced mRNA expression (Fig. 4). However, cells intermediate between chief and oxyphil cells by light microscopy had varying degrees of mRNA expression. Reactivity for PTH mRNA was confined to parathyroid tissue. Adjacent thyroid tissue, where present, was consistently negative. P T H mRNA in relation to pathology Normal--In normal parathyroid tissue, a consistent pattern was seen. mRNA expression was confined to a small proportion of cells, commonly seen in small groups (Fig. 5). Hyperplasiu-The striking difference from normal parathyroid was the proportion of cells showingmRNA production. In some examples, confluent areas of tissue were strongly stained, with the great majority of cells showing activity. The level of expression of any individual cell was, however, no greater than that seen in normal parathyroid. Twelve out of the 22 cases had a distinctly nodular pattern

and there was frequently concordance of expression within individual nodules (Fig. 6). Both examples of parathyroid tissue from known cases of multiple endocrine neoplasia type I showed extensive oxyphil change and minimal mRNA production. No case of water-clear hyperplasia was present in the series. Adenornu-Again, in contrast to normal parathyroid, the proportion of cells expressing mRNA was greatly increased. Uniformity of expression of confluent areas was much less noticeable than in hyperplasia, but only one case had a markedly nodular pattern. Fourteen out of the 23 cases had a detectable ‘rim’ of uninvolved parathyroid tissue. mRNA expression in this rim showed a similar pattern to that of normal parathyroid tissue (Fig. 7). Occasionally a greater proportion of cells showed mRNA expression more comparable to that seen in the adenoma. Carcinoma-Expression of mRNA was seen in all four carcinomas, but to a varying degree and was often markedly focal (Fig. 8). Expression was more variable than that seen in hyperplasia or adenoma. Of the tumours examined, those with overtly invasive features in general had less mRNA activity.

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Fig. 5-Normal parathyroid tissue. Expression of PTH mRNA is mainly seen in small groups of contiguous cells

Fig. &Nodular parathyroid hyperplasia. There is concordance of expression of PTH mRNA throughout the cells of individual nodules

Fig. 7-Moderate PTH mRNA expression in a parathyroid adenoma (left) compared with a pattern of reactivity similar to that seen in normal parathyroid tissue (right)

Fig. &Strong focal expression of PTH mRNA in a parathyroid carcinoma

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therefore, that resting chief cells initially on activation show enlargement of the nucleus and develop Recent advances in non-radioactive in situ cytoplasmic mRNA expression. As synthesis and hybridization techniques have enabled the demon- secretion occur, vacuolation develops. This is prestration of specific mRNA in tissue sections. In sumably then followed by a further resting stage. It this study, we examined the relationship between is known that changes in the chief cell can occur PTH mRNA expression and chief cell morphology, rapidly. Initially, on stimulation chief cells release .~~ as has been and also compared the findings in parathyroid stored p a r a t h ~ r m o n e Subsequently, abnormalities. PTH mRNA has previously been shown experimentally, PTH mRNA is synthesized extracted from parathyroid tissue and demon- within 48 h, the direct regulatory factor being that of strated by Northern blotting,” but the development the ambient calcium Oxyphil cells by this technique have greatly of in situ methods has enabled a study of the cellular reduced or absent mRNA in their fully developed localization of PTH mRNA expression. An assessment of chief cell synthetic activity has form and hence would have little or no synthetic previously largely depended on ultrastructural function. This supports electron microscopy studies studies.’.’’ These have relied on the degree of which have shown poorly developed secretory development of the organelles associated with syn- organelles’ and sparse or absent secretory granules.’ thesis-endoplasmic reticulum, Golgi apparatus, Reports of ‘functioning’ oxyphil lesions (adenoare almost certainly due to the presence of etc. Detection of stored parathormone is technically mas)25’26 difficult and the quantity of stored hormone has cells intermediate between chief and oxyphil cells been shown not to correlate with the rate of cell which do retain some mRNA production. One synthesis.” mRNA production, however, is known hypothesis regarding oxyphil cells which can now be to be more closely correlated with hormone synthe- discarded is that the large number of mitochondria sis with some qualifications related to mRNA with the associated high levels of oxidative energy degradation.20The presence and semi-quantitation represents some form of post-transcriptional of PTH mRNA by this technique therefore offer synthetic block. The technique offers the opportunity of evalua more direct assessment of chief cell synthetic ating mRNA production in a range of functional activity in tissue sections. The expression of PTH mRNA in chief cells and pathological conditions of the parathyroid. varies with cell morphology. This concords with a Firstly, in normal parathyroid, the pattern of accycle of synthetic activity, as suggested by electron tivity in focal small groups of cells raises questions microscopy studies.’*The evidence from the present of the control of cellular activity. Clearly not all study suggests the following. Chief cells with small the cells are responding equally to the ambient dark nuclei and scanty cytoplasm have little or no calcium level. A similar finding is seen in thyroid mRNA expression and therefore correlate with a tissue immunostained for thyroxine, where tall resting state. The strong expression seen in chief columnar cells show strong reactivity whereas adcells with larger, more vesicular nuclei indicates an jacent follicles with flat epithelium are negative active phase. Both vacuolated and non-vacuolated (personal observation). Again, other factors than forms are seen. The nature of vacuolation is un- the TSH level must be operative. This raises the certain, but appears to be an artefact of formalin possibility in both thyroid and parathyroid of fixation and paraffin processing. Comparable some other mechanism such as local paracrine vacuolation is not seen in electron microscopy control. It was of particular interest to compare the findstudies and is much less conspicuous in frozen sections (personal observation). This may reflect the ings in hyperplasia and adenoma. In both, the striklarge number of small vacuoles which characterize ing difference from normal parathyroid tissue was the active synthetic phase.” It has previously been the greatly increased proportion of cells expressing suggested that vacuolated (light) cells are inactive.2’ mRNA. This clearly correlates with the increased However, they are known to be more common in PTH production. Individual cells showed no greater hyperplastic tissue22and this study confirms their maximal mRNA staining than that in normal tissue, active nature. This evidence taken together, there- suggesting that the increased parathormone producfore, suggests that the vacuolated (light) cells of tion is due to the increased amounts of tissue present paraffin processing follow the non-vacuolated stage combined with the much greater proportion of cells and reflect an actively synthetic phase. It appears, active. Previous studies have indicated that the DISCUSSION

PARATHORMONE mRNA EXPRESSION I N PARATHYROID TISSUE

degree of hyperparathyroidism correlates with the total amount ofparathyroid tissue present.22Therim of tissue peripheral to the nodule in adenoma had in most cases a pattern similar to or more active than normal parathyroid tissue. Marked suppression, as might be expected, was not found, It is unclear why this should be the case. One possibility would be the release of some influencing factor from the adenoma itself. It would be useful to compare the rim with uninvolved parathyroid tissue removed at the same time to test this hypothesis. Relationship to ambient calcium level could also be explored. The biological nature of adenomatous change has long remained enigmatic, partly as a result of the lack of clear histological separation of adenoma from hyperplasia. This has led to a search for evidence of clonality in adenoma. An initial report suggested a polyclonal origin as shown by heterozygosit for glucose 6-phosphate dehydrogenase (G6PD).' However, this has now been called into question by two recent studies. Specific restriction fragment length alterations and polymorphism for an x-linked gene have been shown in a series of adenomas which were absent in cases of hyperplasia.28Additionally, a chromosome 1 1 abnormality has also been detected in an a d e n ~ m a . Both * ~ of these studies strongly suggest a monoclonal origin for adenomas and support a biological separation from hyperplasia. In our study, probing for PTH mRNA showed no specific morphological distinction. However, the concordance of expression within nodules, where well formed, in hyperplasia is a prominent feature, not shared by adenomas. Of the four examples of parathyroid carcinoma examined, all showed some degree of synthetic activity. Carcinomas are regularly associated with hyperparathyr~idism,~~ but some tumours are nonfunctional. Of interest in our study was that the more overtly invasive tumours showed less expression, which could correlate with the poorer prognosis seen in the less functional t u m ~ u r s . ~ ' In conclusion, we have shown that PTH mRNA can be satisfactorily demonstrated in routinely processed parathyroid tissue. Morphological differences are seen between active and inactive chief cells, and the patterns of expression in different physiological and pathological states of parathyroid have been explored. Further studies are needed to examine in more detail the relationship between the apparent function state of the chief cells, and hormone storage, secretion, and the physiological state of PTH levels and calcium homeostasis.

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ACKNOWLEDGEMENTS

This study was supported by British Biotechnology Limited and the Trent Regional Health Authority. We are grateful for the photographic assistance of Ms B. Ferrari. The secretarial assistance of Mrs Pauline Coekin, Mrs Wendy Pitts, and Mrs Beverley Richardson is gratefully acknowledged. The assistance of Dr A. MacDonald and Mrs L. Potter is also acknowledged. REFERENCES I . Futrell JM, Roth SI, Sendy PCS, Habener JF, Segre GV, Potts JT. Immunocytochemical localization of parathyroid hormone in bovine parathyroid glands and human parathyroid adenomas. Am J Pathol 1979; 9 4 615422. 2. Pesce C, Tobia F, Carli F, Antonlotti G. The sites of hormone storage in normal and diseased parathyroid glands; a silver impregnation and immunohistochemical study. Histopathology 1989; 15: 157-166. 3. Roth SI, Gallagher MJ. The rapid identification of 'normal' parathyroid glands by the presence of intracellular fat. Am J P a t h d 1976; 84: 521-528. 4. Saffos RO, Rhatigan RM, Urgulu S. The normal parathyroid and the borderline with early hyperplasia; a light microscopic study. Histoparhology 1984; 8: 407422. 5. Capen CC. Fine structural alterations of parathyroid glands in response to experimental and spontaneous changes of calcium in extracellularfluids. Am JMed1971;5 0 598-611. 6. Roth SI. The ultrastructure of primary water-clear cell hyperplasia of the parathyroid glands. A m J Pathol1970; 61: 233-248. 7. Pringle JH. Isotopic or chromogenic? A comparative appraisal of probe labelling for in situ hybridization. Trans R Micros SOC1990; 1: 695-700. 8. Pringle JH, Primrose L, Kind CN, Talbot IC, Lauder I. In situ hyhridization demonstration of poly-adenylated RNA sequences in formalinfixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe. J Patho/ 1989; 158: 279-286. 9. Cox KH, DeLeon DV, Angerer LM, Angerer RC, Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Bioll984; 101: 485-502. 10. Pringle JH, Ruprai AK, Primrose L, er al. In situ hybridization of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten-labelled oligonucleotide probes. J Pathol 1990; 162: 197-207. 1 I . Beaucage SL, Caruthers MH. Deoxynucleoside phosphoramidites-a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett 1981; 2 2 1859-1862. 12. Hendy GN, Kronenberg HM, Potts JT, Rich A. Nucelotide sequence ofcloned cDNAsencoding human preproparathyroid hormone. Proc Nut/ Acad Sci USA 1981 ;78:7365-7369. 13. McBride LJ, Eadie JS, Efcavitch JW, Andrus WA. Base modification and the phosphoramadite approach. Nucleosides Nucleotides 1987; 6 297-300. 14. Deng G, Wu R. An improved procedure for utilizing terminal transferase to add homopolymers to the 3' termini of DNA. Nucleic Acids Res 1981;941734188. 15. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources rich in ribonuclease. Biochemistrv 1979; 18: 52945299. 16. Freeman GJ, Clayberger C, Dekruyff R, Rosenblum DS, Cantor H. Sequential expression of new gene programs in inducer T-cell lines. Proc Nut/ AcadSci U S A 1983; 8 0 40944098. 17. Baba H, Kishihara M, Tohmon M, et al. Identification of parathyroid hormone messenger RNA in an apparently non-functioning parathyroid carcinoma transformed from a parathyroid carcinoma with hyperparathyroidism. J CIin Endocrinol Metab 1986; 6 2 247-252.

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