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Department of Laboratory Medicine (L.S.), St. Michael’s Hospital, University of Toronto, Toronto, Ontario, M5B 1W8, Canada; Genentech, Inc. (L.P.-B., W.W.), Cardiovascular Research, South San Francisco, California, and Department of Physiology (V.C., A.B.), School of Medicine, Southern Illinois University, Carbondale, Illinois 62901-6512
Address all correspondence and requests for reprints to: Lucia Stefaneanu, Ph.D., Department of Pathology, St. Michael’s Hospital, 30 Bond Street, Toronto, Ontario, M5B 1W8, Canada. E-mail: L.Stefaneanu{at}utoronto.ca
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Abstract |
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Introduction |
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Many tissues of epithelial and mesenchymal origin produce IGF-I during both fetal and adult life (3). IGF-I was found to be mitogenic and induce terminal differentiation of many cell types. The interaction of IGF-I with its receptor is mediated by high affinity IGF-I binding proteins (4). IGF-I acts through the type I IGF receptor, which is a membrane tyrosine kinase homologous to the insulin receptor (5).
Pituitary production of IGF-I and its binding proteins was demonstrated first in tissue culture of rat explants (6, 7). IGF-I messenger RNA (mRNA) was found in rat pituitary by Northern analysis (8), and by in situ hybridization (9). It was also shown that a rat pituitary tumor cell line (GH3) secreting GH and PRL synthesizes IGF-I (10) consistent with the view that IGF-I is probably produced by pituitary somatotrophs. However, by in situ hybridization IGF-I RNA was diffusely distributed throughout the anterior lobe (9, 11) suggesting that intrinsic IGF-I may affect other cells besides those producing GH.
The role of pituitary IGF-I has yet to be elucidated. It is evident that pituitary IGF-I contributes to the inhibition of GH synthesis and release (12). Another possible role of pituitary IGF-I is the mediation of proliferative effects of estrogen on lactotrophs as suggested by the fact that estrogen administration to ovariectomized rats increased adenohypophysial content of IGF-I and IGFBP-2 RNAs (11).
Mice with null mutations of IGF-I gene were produced by disruption of the gene at amino acid 50 (13) or amino acid 15 (14). The mice with disrupted IGF-I gene (IGF-I knockout mice) have at birth a 60% reduction of body weight compared with wild-type mice and approximately 84% or 95% of pups die perinatally. The primary change in newborns is severe hypoplasia of muscles, especially in heart, diaphragm and tongue. Body weights of the surviving adults show 30–45% decrease compared with wild-type siblings. The dwarfs are infertile and show multiple abnormalities of reproductive system (15). These data indicate that IGF-I is important for mouse viability, plays a major role in fetal development and is essential for both fetal, postnatal body growth, and fertility.
In the present study, we report the pituitary morphologic changes in a line of mice with disrupted IGF-I gene. The results indicate that in the absence of IGF-I, structural and functional alterations occur in somatotrophs and lactotrophs, and IGF-I treatment reverses in part such changes.
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Materials and Methods |
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Treatments
Two-month-old male and female Igf1-/-
and wild-type mice were daily injected sc with 10 mg/kg human
recombinant IGF-I. Control IGF-I knockout and wild-type mice of same
age and sex received excipient. Body weights were measured every day,
and the amount of injected IGF-I was adjusted accordingly. After 2
weeks, the mice were killed by CO2 inhalation, the blood
was collected, and plasma was frozen and stored at -70 C till used for
PRL RIA.
Light microscopy
Paraffin sections of 5 µm thickness were stained with
hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) technique.
Immunocytochemistry
The streptavidin-biotin peroxidase complex method of Hsu
et al. (16) using a kit (LSAB, DAKO Corp.,
Carpinteria, CA) was applied. The primary antibodies included antirat
GH (diluted 1:1760), rat PRL (1:800), rat ßTSH (1:500), rat ßFSH
(1:250), rat ßLH (1:800) and human ACTH (1:1200) (all kindly donated
by Dr. A. F. Parlow, National Institute of Diabetes and Digestive
and Kidney Diseases, Bethesda, MD). All antibodies were polyclonal
raised in rabbit except for GH raised in monkey. The reaction product
was visualized using as chromogen diaminobenzidine. Negative controls
were performed by replacing the primary antibodies with phosphate
buffer saline. The proliferation nuclear antigen (PCNA) was
demonstrated using a monoclonal antibody (DAKO Corp.;
PC10) diluted 1:70.
Cells immunoreactive for GH, PRL, ACTH, and LH were counted with a 40x objective lens within a 0.01-mm2 ocular grid. The total number of positive cells was expressed as percentage of total number of cells within the same area. The counting covered the horizontal section of a pituitary in every case, and comprised approximately 2,500–5,000 cells/section. The diameter of GH immunoreactive cells was measured on microphotographs taken with 100x objective; two diameters were measured for 30–40 cells.
In situ hybridization
In situ demonstration of GH-, PRL-, and POMC-mRNAs
was performed on 5 µm deparaffinized sections applying
oligodeoxynucleotide probes corresponding to amino acids 145–151 of
mouse GH, 64–70 of mouse PRL and 99–108 of rat POMC. The probes were
labeled by the 3'-end method with 35S-dATP using a kit
(NEP-100, DuPont Canada, Mississauga, Ontario, Canada) and
purified with NENSORB-TM cartridge included in the kit. The details of
hybridization conditions, and authoradiography were described elsewhere
(17). Negative controls included: a) RNase predigestion (100 µg/ml;
Sigma Chemical Co., St. Louis, MO) of tissue sections, on
which the hybridization protocol was carried out; b) Competitive
hybridization assay with 100-fold excess of unlabeled probe added to
the labeled probe. In all negative control sections, the signal was
effectively abolished to up to 5 silver grains/cell (attributable to
nonspecific binding of the probes to DNA or other subcellular
elements).
A quantitative evaluation of signal intensity was performed for GH and PRL mRNAs, by counting silver grains with a 100x-oil objective over 50–80 adenohypophysial cells/section. Nonspecific hybridization was obtained from the number of silver grains on intermediate and posterior lobes (1–5 silver grains/cell), and the mean number was subtracted from the mean number of silver grains on anterior lobe cells.
Transmission electron microscopy
Pituitaries fixed in glutaraldehyde were postfixed in 1% osmium
tetroxide, dehydrated and embedded in Epon-Araldite mixture. Semithin
sections were stained with toluidine blue and ultrathin sections with
uranyl acetate and lead citrate, and investigated with a Philips 419LS
transmission electron microscope.
RIA
The levels of plasma PRL were measured by RIA using antibodies
against mouse PRL and purified mouse PRL preparations for iodination
and standards as described previously (18).
Statistical analysis
Data were evaluated statistically by the one-way ANOVA, followed
by two-pair test using Bonferroni correction. Results were expressed as
mean ± SE.
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Results |
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In both homozygous and heterozygous mice, the pituitaries contained
GH-, ACTH-, TSH-, FSH-, and LH-immunoreactive cells with the same
pattern of disposition and size as seen in control pituitaries. No PRL
immunoreactive cells were present in any glands at this age. Cell
counts revealed no differences between the percentages of GH-, ACTH-,
TSH-, and LH-cells in the pituitaries of
Igf1-/- and Igf1I+/+
mice (Table 1
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). PRL RNA was not detected in any
group. POMC RNA had the same intensity of hybridization signal in all
three groups.
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Adult pituitaries
Homozygous mice. The pituitaries of
Igf1-/- mice were decreased in weight
approximately proportionally with their body weights in comparison to
wild-type siblings (Table 2). On H&E
sections, the anterior lobes contained numerous acidophils interspersed
with basophils and chromophobes as seen in wild-type pituitaries. In
female mice, the acidophils appeared to be smaller than in controls.
Intermediate and posterior lobes showed no morphologic changes.
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), (Fig. 2). In males, the signal for PRL mRNA was
slightly decreased by 20%, whereas in females, the decrease was more
pronounced (53% of control value) (Fig. 3). The relative number of GH
immunoreactive cells was higher than in wild-type mice and reached
significance in females. Considering the decrease in the number of PRL
immunoreactive cells, an apparent increase in the percentage of GH
cells was anticipated. In female mice, the diameter of GH
immunoreactive cells was significantly smaller, measuring approximately
80% of that of wild-type ones (Fig. 4, a
and b). The signal for GH RNA was augmented by about 50% in both
genders compared with wild-type mice (Table 4). In females, considering the decrease
in size of the GH cells, the increase in the GH RNA signal was in
reality less than 50%. The relative percentage of ACTH-, TSH-, and LH-
immunoreactive cells was slightly increased, due to the decrease of PRL
immunoreactive cells. In both sexes of knockout mice the intensity of
silver grains signaling POMC RNA did not differ from wild-type
mice.
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). Male pituitaries contained
somatotrophs with some features of stimulation. The slightly eccentric
nuclei with occasional indentations contained one or two nucleoli. In
some somatotrophs, the rough endoplasmic reticulum (RER) was very
prominent composed of parallel arrays of cisternae (Fig. 6). In a few cells, accumulation of large
deposits of secretory material in the intercellular space and extruded
secretary granules toward capillaries were seen. The size of
somatotrophs in IgfI-/- males was comparable
with that of normal pituitaries. In the
IgfI-/- female mouse, the cytoplasmic area of
somatotrophs was reduced (Fig. 5), and the RER was prominent in some of
them. Occasionally, extrusion of secretory granules was evident (Fig. 7). No changes in the ultrastructural
features of corticotrophs, thyrotrophs and gonadotrophs were evident in
both genders.
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), no adenohypophysial morphologic changes were
detected in heterozygous mice. The percentages of hormone-containing
cells were similar to those found in control pituitaries (Table 3).
There were no differences in the intensity of silver grains coding for
GH, PRL, and POMC mRNAs between Igf1+/- and
Igf1+/+ mice.
Effect of IGF-I treatment. Two weeks treatment with
physiologic doses of IGF-I resulted in significantly increased body
weights of Igf1-/- and modest increase of
Igf1+/+ mice (Table 4). The pituitary weights
were slightly increased, without reaching significance in both sexes of
Igf1-/- mice.
In both genders of IGF-I knockout mice, the basal levels of plasma PRL
were elevated compared with wild-type mice (Table 4), despite the
reduced number of pituitary PRL immunoreactive cells. IGF-I
administration to wild-type mice induced a rise of plasma PRL, which
reached significance in females, that was accompanied by an increase of
pituitary PRL RNA signal (Table 4), and no change in the number of PRL
immunoreactive cells. In Igf1-/- mice IGF-I
treatment had no effect on mean plasma PRL levels (Table 4). IGF-I did
not restore the number of PRL immunoreactive cells, and had no effect
on the intensity of PRL RNA signal in both sexes of IGF-I knockout mice
(Table 4).
The IGF-I administration did not restore the size of GH immunoreactive
cells in female Igf1-/- mice (Fig. 4c), but
induced a modest, statistically not significant increase in
wild-type females (Fig. 4d). In both genders of IGF-I
knockout mice and wild-type mice, the GH hybridization signal
was decreased by about 40%. No effect on the number of any
hormone-containing cells was found.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the ultrastructural features of stimulation, the somatotrophs of IgfI-/-mice were not hypertrophied, contrary in females their cytoplasmic area was reduced by approximately 20% compared with normal mice. It is known that different mechanisms govern the size of a cell and its multiplication. The present findings suggest the involvement of IGF-I in the control of cell size in a gender-specific fashion. Protracted stimulation by GHRH, as seen in transgenic mice overexpressing GHRH and GHRH-treated rats, leads to hypertrophy and hyperplasia of somatotrophs (22, 24). In the absence of IGF-I, we assume that in females the somatotrophs did not reach the normal size. The fact that IGF-I administration did not restore the size of GH immunoreactive cells reveals the importance of locally produced IGF-I in this process.
Unexpectedly, in fetal pituitaries (e18.5) the level of GH RNA was decreased by more that 50% in both Igf1-/- and Igf1+/- mice. Serum IGF-II levels are highest during fetal life and decline rapidly after birth in contrast with serum IGF-I levels, which are low in the fetal and postnatal periods compared with adult levels (1). In the normal fetus, it can be assumed that IGF-I receptors are occupied by IGF-I, due to their higher affinity for IGF-I than for IGF-II. In the absence of IGF-I, or very low IGF-I levels it may be that IGF-II binds the unoccupied IGF-I receptor and inhibits pituitary GH gene transcription. Alternatively, the decreased GH RNA signal in homozygous and heterozygous mice may reflect a higher rate of utilization/degradation or a shorter half-life of the message. Because no changes were depicted in the percentage of GH-, ACTH-, TSH-, and LH- immunoreactive cells and ultrastructural features of adenohypophysial cells, it can be concluded that IGF-I does not play a role in the differentiation of pituitary cell types.
Remarkable changes occurred in the lactotrophs of mice with disrupted IGF-I gene. PRL immunoreactive cells were significantly reduced in number and contained a weaker signal for PRL RNA. Despite these signs of inhibition, the basal plasma PRL levels were elevated without reaching significance in both genders of Igf1-/- mice. IGF-I administration raised significantly the plasma PRL levels in wild-type mice. The change was accompanied by enhanced pituitary signal for PRL RNA. In both genders of knockout mice neither plasma PRL, nor the intensity of PRL mRNA signal differed from untreated controls. It can be speculated that these unexpected findings are due to: 1) the lack of the intrinsic and/or circulating IGF-I; 2) disturbed gonadal and adrenal steroidogenesis; both 1) and 2).
Conflicting results are available on the in vitro effect of IGF-I on lactotrophs, and no in vivo studies were reported to our knowledge. An earlier in vitro study using semipurified preparation of IGF-I reported the inhibition of PRL secretion of rat pituitary (25). Others showed no effect on normal pituitary cells, or GH3 cell line (12, 26, 27), or stimulation of PRL secretion by cultured pituitaries of normal rat and human pituitary adenoma cells (28, 29). In vitro experiments on rat pituitary cells suggested that stimulation of PRL release by IGF-I is mediated by VIP (30). Contrary, in GH4C1 cells, IGF-I induced significantly PRL gene transcription through sequences located in the proximal promoter without affecting GH gene (27). Our results in normal mice support the stimulatory role of IGF-I on PRL release and accumulation of PRL transcripts. It is possible that in vivo, IGF-I induces PRL gene transcription, as seen in GH4C1 pituitary cell line. The lack of response of pituitary lactotrophs to IGF-I in null mutant mice supports the role of locally produced IGF-I in PRL stimulation. Because somatostatin has an inhibitory effect on PRL release (31), in knockout mice it is possible that the decreased somatostatin inhibition affected both somatotrophs and lactotrophs. Whether circulating or hypothalamic IGF-I influences hypothalamic dopamine secretion and/or release remains to be studied. The higher basal levels of plasma PRL reflects most probably decreased tonic inhibition of lactotrophs.
It can be also speculated that the inhibition of PRL cells is due to
decreased circulating levels of estrogen and testosterone, well-known
stimulators of PRL secretion in females and males, respectively. That
IGF-I mediates some of the estrogen effects was suggested by a study in
which the steroid administration to ovariectomized mice increased
pituitary IGF-I RNA (11). Baker et al. (15) investigated the
gonads of another line of IGF-I knockout mice and found delayed
maturation of Leydig cells, and 70% reduction of plasma testosterone.
The ovaries contained follicles in different phases of maturation but
no corpora lutea or corpora albicantia indicating the lack of
ovulation. The serum concentration of estradiol in females was about
53% of the normal value. Although decreased, this level of estradiol
does not justify the marked reduction in number of lactotrophs.
Moreover, we found gonadotrophs in normal range and no "gonadectomy
cells." Applying in situ hybridization for 
estrogen
receptor (ER) mRNA we found no differences in the intensity of
signal in pituitaries of Igf1-/- compared with
control pituitaries (results not included).
The role of IGF-I in the stimulation of PRL secretion is supported by the findings in dwarf rats with isolated GH deficiency, in which pituitary and serum PRL levels, PRL mRNA and the number of lactotrophs are markedly reduced (32, 33). In PEPCK/bGH transgenic mice with very high levels of foreign GH, we reported PRL cell hyperplasia in females (34). Plasma testosterone level is normal in PEPCK/bGH mice, whereas in females the estrous cycle is significantly longer, and in those females that are fertile, the number of ova shed is significantly greater than in normal females, indicating hyperestrogenization (35). Based on our results in normal and IGF-I knockout mice, as well as those in dwarf rats and transgenic mice, we suggest that IGF-I interacts with estrogen/ER pathway to stimulate PRL cells.
The mice with mutated IGF-I gene resemble patients with Laron-type dwarfism. The children present severe growth retardation and despite the high levels of GH, the circulating IGF-I levels are very low due to mutations of GH receptor gene (36). Both children and adults with Laron’s syndrome have elevated basal PRL levels and IGF-I treatment for 2 months does not normalize this parameter (37). Mice with inactive GH receptor have also significantly elevated PRL levels and undetectable plasma IGF-I, further supporting the role of IGF-I in regulation of PRL secretion (38).
In conclusion, the present study confirms the role of circulating IGF-I in the inhibition of GH cells, and reveals for the first time the role of pituitary IGF-I in the control of the size of this cell type, in a gender specific manner. Whereas in normal mice, IGF-I stimulated the lactotrophs, no such effect was found in mice with inactive IGF-I in which the lactotrophs were inhibited. The inability of IGF-I to stimulate the lactotrophs may be due to the absence of pituitary IGF-I, and to decreased levels of sex hormones. The results suggest that in pituitary, the locally produced IGF-I cross-talk with ER to elicit a stimulatory effect on lactotrophs.
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Acknowledgments |
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Footnotes |
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Received February 8, 1999.
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References |
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