NOEMI E. LOSINSKI, NILS BILLESTRUP, JENNY PRICE, AND WYLIE VALE. Department of Pathology, St. Michael's Hospital, University of Toronto (L.S., K.K., ...
0013-7227/89/1255-2710$02.00/0 Endocrinology Copyright © 1989 by The Endocrine Society
Vol. 125, No. 5 Printed in U.S.A.
Adenohypophysial Changes in Mice Transgenic for Human Growth Hormone-Releasing Factor: A Histological, Immunocytochemical, and Electron Microscopic Investigation* LUCIA STEFANEANU, KALMAN KOVACS, EVA HORVATH, SYLVIA L. ASA, NOEMI E. LOSINSKI, NILS BILLESTRUP, JENNY PRICE, AND WYLIE VALE Department of Pathology, St. Michael's Hospital, University of Toronto (L.S., K.K., E.H., S.L.A., N.E.L.), Toronto, Ontario, M5B 1W8 Canada; Hagedorn Research Laboratory (N.B.), Gentofte, Denmark DK-2820; and Biotechnology/Industrial Associates, Inc. (J.P.), and the Clayton Foundation Laboratories for Peptide Biology (W. VJ, the Salk Institute, La Jolla, California 92037
pituitaries and cells resembling human adenomatous mammosomatotrophs in the other three glands. All of these cells, regardless of their ultrastructural features, contained secretory granules heavily labeled for GH by immunogold technique; PRL labeling varied from cell to cell, with the predominance of a weak immunostaining and was colocalized with GH in secretory granules. These results indicate that chronic exposure to GRF excess leads to mammosomatotroph hyperplasia. It is suggested that GH cells proliferate and transform to mammosomatotrophs in response to GRF stimulation. Focal PRL cell hyperplasia noted in three pituitaries could also be due to a GRF effect. Longer exposure to GRF is needed to clarify whether GRF can cause adenoma. {Endocrinology 125: 2710-2718,1989)
ABSTRACT. The effect of protracted GH-releasing factor (GRF) stimulation on adenohypophysial morphology was investigated in six mice transgenic for human GRF (hGRF). All animals had significantly higher plasma levels of GH and GRF and greater body weights than controls. Eight-month-old mice were killed, and the markedly enlarged pituitaries were studied by histology, immunocytochemistry, electron microscopy, and immunogold method, using double labeling at ultrastructural level. In all pituitaries, a massive hyperplasia, chiefly of mammosomatotrophs, was found. These bihormonal cells, containing GH and PRL, were demonstrated by light microscopy and ultrastructural immunocytochemistry. Electron microscopy revealed the presence of cells with characteristics of GH cells in three
T
HE STUDY of transgenic animals represents a novel approach to obtain a deeper insight into the pathogenesis and development of disease. In such animals, inclusion of an extra gene into the genome can induce various pathological states (1, 2). Insertion of hormone genes can elicit different endocrine abnormalities, allowing for the investigation of a protracted effect of various hormones on the endocrine system. Various hypersecretory states similar to those occurring in human pathology can be reproduced. These disease models can shed light on the pathophysiology of endocrine disorders and help to better understand the sequence of events leading to endocrine disease. It has been shown that metallothionein (MT)-human GH (hGH) fusion genes cause prolonged oversecretion Received June 8,1989. Address requests for reprints to: Dr. Lucia Stafaneanu, Department of Pathology, St. Michael's Hospital, University of Toronto, Toronto, Ontario, M5B 1W8 Canada. * This work was supported by Grant MT-6349 from the Medical Research Council of Canada, the Clayton Foundation for Research, California Division, and Philips Petrolium.
of GH and stimulate body growth of mice (3). Murphy et al. (4) found that inclusion of vasopressin-SV40 oncogene in the genome of mice results in the formation of tumors of endocrine pancreas and adenomas of the pituitary. Recently, mice transgenic for GH-releasing factor (GRF) have been developed (5), offering a model to study the protracted effect of GRF on its target, the pituitary gland (6). It has been shown that the pituitaries of mice expressing MT-GRF gene are increased in weight and size, due to hyperplasia of GH cells (somatotrophs), as demonstrated by light microscopic immunocytochemistry (6). In the present study, complex morphological investigation of the adenohypophyses of MT-GRF transgenic animals, including immunocytochemistry and electron microscopy, demonstrated the presence of a massive hyperplasia of mammosomatotrophs. These cells containing GH and PRL were identified by light and electron microscopic immunocytochemistry; ultrastructurally, they differed from mammosomatrophs found in normal mouse pituitary.
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PITUITARY MORPHOLOGY IN hGRF TRANSGENIC MICE mMT-1 PROMOTOR
SstI
Hindi
hGRF
Pst I
SV-40
SMALL T
small t fusion gene used for injection is shown in Fig. 1. A 713basepair (bp) fragment of the mouse MT-I promoter, containing elements responsible for metal induction and transcription initiation, is fused to 220 bp of the human GRF gene, encoding the NH2-terminal 30-amino acid signal peptide and the 40amino acid form of hGRF. The polyadenylation signal was provided by fusion to a 847-bp fragment of the SV40 virus small t poly (A). At weaning, tail DNA of offsprings was analyzed for the presence of hGRF by DNA dot-blotting method. From 6 weeks of age, the transgenic animals and six age- and sex-matched controls were maintained on water containing 25 nM ZnSO4 and laboratory chow ad libitum. Blood was collected from the tails of 8-week-old animals to measure serum GH and GRF
POLY A
Bam HI
Sst I 200 bp
FIG. 1. hGRF/mouse MT-I/S40 small t fusion gene construct.
Materials and Methods Six transgenic mice (three females and three males) containing the MT-hGRF fusion gene were produced as described by Hammer et al. (5). Human GRF (hGRF)/mouse MT-I/S40
TABLE 1. Body weights, pituitary weights, and GRF and GH blood levels of transgenic mice (T) compared to those of control mice (C) BW(g) Case no. and sex l.F
2, F 3, F 4, M 5, M 6, M Mean ±SEM
Pituitary wt (mg)
mGH (ng/ml)
T
C
T
C
T
C
T
C
39.4 41.9 37.2 42.4 44.1 41.0
23.7 21.2 20.8 27.8 26.7 27.1
6.7 9.8
0.9 1.0 0.9 0.8
