Path. Res. Praet. 183, 577-579 (1988)

Analysis of Mammosomatotropic Cells in Normal andNeoplastic Human Pituitaries*

Ricardo V. LloydDepartment of Pathology, University of Michigan Medical Center, Ann Arbor, USA

SUMMARY

The reverse hemolytic plaque assay (RHPA) was used to analyze hormone secretion innormal and neoplastic human pituitary cells. Immunocytochemical (ICC) staining forPRL and GH with the peroxidase method and ultrastructural ICC with colloidal goldlabeling were used along with the RHPA to analyze for mammosomatotropic (MS) cells inthese dissociated and cultured pituitary cells. MS cells were identified in both normal andneoplastic pituitary tissues. Quantitation of plaque areas, showed that PRL cells fromnormal pituitaries had significantly larger plaque areas than PRL cells from PRL-produc­ing adenomas or from mixed PRL-GH producing adenomas.

Introduction

Although GH and PRL are produced by morphologi­cally distinct cell types, recent evidence has indicated thatsome cells in rat and human pituitaries produce both PRLand GH1,3-12. The reverse hemolytic plaque assay(RHPA) and immunocytochemical techniques (ICC) haveprovided simple and direct methods to analyze pituitarytissues for mammosomatotropic (MS) cells.

In this study we used a combination of the RHPA, ICCand ultrastructural immunohistochemistry to analyze forMS in normal human pituitaries obtained at autopsy andsurgically excised PRL- and GH-producing human pitu­itary tumors. In addition the plaque areas produced bycells after the RHPA, which is an index of hormone secre­tion, was analyzed in normal and neoplastic humanpituitaries.

Material and Methods

Pitutiary tissues obtained during postmortem examination andfrom surgically excised adenomas were dissociated with trypsinand used for cell cultures as previously described for rat

• This work was supported in part by NIH Grants CA 42951and 37238 and Council for Tobacco Research Grant No 1856.

pituitaries9• The RHPA and ICC staining were performed as pre­viously described9

• In some assays a combination of the RHPAand ICC staining was carried out on the same slides. Ultrastruc­tural ICC with two particle sizes of immunogold reagents wereused2• Sections were labeled with antibodies to human PRL andGH used at a 1/1000 dilution for 60 min. Goat anti-rabbit 15 nmand 50 nm gold particles, diluted 1/10, were used to identify PRLand GH positive secretory granules. The specificity of immu­nolabeling was tested by absorbing each antiserum with itsrespective antigen and by incubating GH antiserum with 10 It!I ofPRL and PRL antisera with 10 I1g of GH before immunolabeling.

Quantitation of Plaque Areas

Quantitation of plaque areas which was related to the amountof secreted hormone1o was done with a Bioquant II with a digitiz­ing morphometry program, an IBM computer, and a Zeiss IImicroscope. Statistical differences between groups were evalu­ated by the Student t test.

Results

Hemolytic plaques were seen after the sequential incu­bation of cells previously cultured for 24 hours withprimarily antibody followed by complement (Fig. 1).Omission of the primary antibody or of the complementdid not produce hemolytic plaques. Likewise substitutionof absorbed PRL or GH for the respective antiserum

578 . R. V. Lloyd

Fig. 1. Reverse hemolytic plaque assay analy­sis of a PRL-producing adenoma with prolac­tin antiserum showing three plaque-formingcells (x 210).

Fig. 2. Ultrastructural immunocytochemical localization of GH and PRL and MS cells from a GH-producing adenoma. The PRLantiserum and GH antiserum were localized with 15 nm and 40 nm gold particles respectively (X 17000).

Mammasomatotropic Cells . 579

Discussion

Table 1. Analysis of plaque areas after the reverse hemolyticplaques assay in normal and neoplastic human pituitary cells

Normal Pituitary 12.983 ± 687 (93) 11.748 ± 730 (91)PRL Adenoma 8.626 ± 812** (294) 10.920 ± 1046 (109)GH Adenoma 8.976 ± 1230 (32) 9.773 ± 380 (61)

** p < 0.01; compared to normal pituitary.n = number of plaques analyzed.Plaque areas were analyzed with a Bioquant II with a digitizingmorphometry program.

References

1 Bassetti M, Spada A, Arosio M, Vallar L, Brina M, Giannat­tasio G (1986) Morphological studies on mixed growth hormone(GH) - and prolactin (PRL) - secreting human pituitary adeno­mas. Coexistence of GH and PRL in the same secretory granule.J Clin Endocrinol Metab 62: 1093-1100

2 Bendayan M (1982) Double immunocytochemical labelingapplying the protein A-gold technique. J Histochem Cytochem30: 81-85

3 Frawley LS, Boockfor FR, Hoeffler JP (1985) Identificationby plaque assays of a pituitary cell type that secretes both growthhormone and prolactin. Endocrinology 116: 734-737

4 Fumagalli G, Zanini A (1985) In cow anterior pituitary,growth hormone and prolactin can be packed in separategranules of the same cell. J Cell Bioi 100: 2019-2024

5 Goluboff IG and Ezin C (1969) Effect of pregnancy on thesomatotropic and the prolactin cell of the human adenohypophy­sis. J Clin Endocrinol 29: 1533-1538

6 Halmi NS (1982) Occurrence of both growth hormone- andprolactin immunoreactive material in the cells of human somato­tropic pituitary adenomas containing mammotropic elements.Virchows Arch [Pathol Anat] 398: 19-31

7 Heitz PU, Landolt AM, Zenklusen H-R, Kasper M, Reubi J­C, Oberholzer M, Roth J (1987) Immunochytochemistry ofpituitary tumors. J Histochem Cytochem 35: 1005-1011

8 Horvath E, Kovacs K, Killinger DW, Smyth HS, Weiss MH,Ezrin C (1983) Mammosomatotroph cell adenoma of the humanpituitary: a morphologic entity. Virchows Arch [Pathol Anat]398: 277-289

9 Lloyd RV, Coleman K, Fields K, Nath V (1987) Analysis ofprolactin and growth hormone production in hyperplastic andneoplastic rat pituitary tissues by the hemolytic plaque assay.Cancer Res 47: 1087-1092

10 Luque EH, Munoz de Toro M, Smith PE, and Neill JD(1986) Subpopulations of lactotropes detected with the reversehemolytic plaque assay show differential responsiveness todORamine. Endocrinology 118: 2120-2124

1 Nikitovich-Winer BM, Atkin J, Maley BE (1987) Colocali­zation of prolactin and growth hormone within specificadenophypophyseal cells in male, female and lactating femalerats. Endocrinology 121: 625-630

12 Zimmerman EA, Defendeni R, Frantz AG (1974) Prolactinand growth hormone in patients with pituitary adenomas: a cor­relative study of hormone in tumor and plasma byimmunoperoxidase technique and radioimmunoassay. J ClinEndocrinol Metab 38: 577-585

appropriate stimulation or (b) they could be a permanentcell type in the pituitary gland. Some studies have shownthat the pituitary of pregnant cows4, rats lO as well as thehuman pituitary during pregnancy5 contained a significantnumber of MS cells suggesting that pregnancy may be astimulus for proliferation of this cell type.

Plaque Area(n) GH (u2) (n)

Plaque AreaPRL (u2)

This study revealed MS cells in normal humanpituitaries with a variety of methods. MS cells had beenidentified in human pituitary neoplasms by several inves­tigatorsl

, 6, 8. The present analysis used a combinationof four techniques including (a) the RHPA, (b) ICC, (c)combined RHPA and ICC and (d) ultrastructuralimmunochemistry to show that a significant number ofPRL and GH producing cells in the human pituitary areMS.

The significance and function of MS cells is not com­pletely understood. These cells could (a) be a precursor cellfor PRL and GH cells and undergo differentiation under

resulted in no plaque formation. The percentage of MScells estimated by this method ranged from 29 to 49% fornormal pituitaries, and from 10 to 42% for three GH­producing adenomas and 15% for two PRL-producingadenomas. The results of the ICC assay were in generalagreement with the RHPA analysis.

When the combined RHPA-ICC analysis was used toanalyze for MS cells the percentage of MS cells by thisdirect approach generally agreed with the independentanalyses by the RHPA and ICC techniques. Ultrastructuralimmunochemical analysis of cultured pituitary cells andpituitary cells analyzed before cell dissociation revealedmixed PRL and GH labeling in the same cells and withinthe same granules (Fig. 2).

Measurements of plaque areas revealed that PRL cellsfrom the normal pituitaries had significantly larger plaqueareas compared to PRL cell areas from PRL-producing orGH-producing adenomas. The plaque areas for GH pro­ducing cells were not significant among the differentgroups.

Group

Received January 30, 1988 . Accepted March 9, 1988

Key words: Reverse hemolytic plaque assay - Immunogold labeling - Pituitary tumors - Mammasomatotropic cells ­Adenoma

Ricardo V. Lloyd, Department of Pathology, University of Michigan Medical Center, 1500 E Medical Center Drive, 2 G332 Box0054, Ann Arbor, MI 48109-0054, USA