IGF-I is synthesized in the human and rat anterior pituitary glands. The present study was designed to clarify the growth-promoting action of IGF-I on mouse pituitary cells in a primary serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of bromodeoxyuridine (BrdU). BrdU labelling in the nucleus was found in all types of secretory cells: corticotrophs, thyrotrophs, gonadotrophs (LH cells and FSH cells), somatotrophs and mammotrophs. IGF-I (75 ng/ml) stimulated the proliferation of corticotrophs and mammotrophs among the pituitary secretory cells. IGF-I receptor mRNA was detected in the cultured pituitary cells using reverse transcription (RT)-PCR, indicating that mouse pituitary cells expressed IGF-I receptors. Insulin (100 ng/ml) or IGF-I (7.5 ng/ml) failed to increase the percentage of BrdU-labelled cells. However, treatment with insulin (100 ng/ml) plus IGF-I (7.5 ng/ml) increased the percentage of BrdU-labelled cells in a synergistic-like manner. Genistein, a tyrosine kinase specific inhibitor, decreased the IGF-I-induced cell proliferation, indicating that IGF-I acts through IGF-I receptors. IGF-I mRNA was also detected in the cultured pituitary cells by RT-PCR, and its peptides were immunocytochemically detected. The present results demonstrate that all types of pituitary secretory cells have the ability to proliferate in our serum-free culture system. IGF-I synthesized in the pituitary gland may stimulate the growth of pituitary cells, in particular corticotrophs and mammotrophs, by an autocrine or paracrine mechanism.

IGF-I is expressed in somatotrophs, and IGF-I receptors are expressed in most somatotrophs and some corticotrophs in the mouse pituitary gland. Our recent study demonstrated that IGF-I stimulates the proliferation of corticotrophs in the mouse pituitary. These results suggested that somatotrophs regulate corticotrophic functions as well as somatotrophic functions by the mediation of IGF-I molecules. The present study aimed to clarify factors regulating pituitary IGF-I expression and also the roles exerted by IGF-I within the mouse anterior pituitary gland. Mouse anterior pituitary cells were isolated and cultured under serum-free conditions. GH (0.5 or 1 microg/ml), ACTH (10(-8) or 10(-7) M), GH-releasing hormone (GHRH; 10(-8) or 10(-7) M), dexamethasone (DEX; 10(-8) or 10(-7) M) and estradiol-17beta (e2; 10(-11) or 10(-9) M) were given for 24 h. IGF-I mRNA levels were measured using competitive RT-PCR, and GH and pro-opiomelanocortin (POMC) mRNA levels were measured using Northern blotting analysis. GH treatment significantly increased IGF-I mRNA levels (1.5- or 2.1-fold). ACTH treatment did not alter GH and IGF-I mRNA levels. IGF-I treatment decreased GH mRNA levels (0.7- or 0.5-fold), but increased POMC mRNA levels (1.8-fold). GH treatment (4 or 8 microg/ml) for 4 days increased POMC mRNA levels. GHRH treatment increased GH mRNA levels (1.3-fold), but not IGF-I mRNA levels. DEX treatment significantly decreased IGF-I mRNA levels (0.8-fold). e2 treatment did not affect IGF-I mRNA levels. GH receptor mRNA, probably with GH-binding protein mRNA, was detected in somatotrophs, and some mammotrophs and gonadotrophs by in situ hybridization using GH receptor cDNA as a probe. These results suggested that IGF-I expression in somatotrophs is regulated by pituitary GH, and that IGF-I suppresses GH expression and stimulates POMC expression at the transcription level. Pituitary IGF-I produced in somatotrophs is probably involved in the regulation of somatotroph and corticotroph functions.

Oestrogen stimulates the proliferation of pituitary cells. The present study was designed to clarify the involvement of transforming growth factor-alpha (TGF-alpha) in the oestrogen-induced growth of mouse pituitary cells in vitro. Anterior pituitary cells obtained from ICR male mice were cultured in a primary serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of bromodeoxyuridine. Secretory cell types were immunocytochemically determined. Treatment with TGF-alpha (0.1 and 1 ng/ml) for 5 days stimulated cell proliferation. Since TGF-alpha binds to the epidermal growth factor (EGF) receptor, this action may be exerted through the EGF receptor. Oestradiol-17beta (OE(2), 10(-)(9) M) stimulated mammotrophic and corticotrophic cell proliferation. RG-13022, an EGF receptor inhibitor, inhibited the cell proliferation induced by EGF or OE(2), showing that the EGF receptor was involved in the growth response in mammotrophs and corticotrophs. Treatment with antisense TGF-alpha oligodeoxynucleotide (ODN) inhibited the cell proliferation induced by OE(2), but treatment with antisense EGF ODN did not. RT-PCR analysis revealed that OE(2) stimulated TGF-alpha mRNA and EGF receptor mRNA expression. These results indicate that TGF-alpha mediates the stimulatory effect of oestrogen on the pituitary cell proliferation in a paracrine or autocrine manner, and that EGF receptor expression is stimulated by oestrogen.

The presence and possible physiological roles of alpha-melanocyte-stimulating hormone (alpha-MSH) in the peripheral tissues of birds have not been established. By a combination of RT-PCR, immunocytochemistry and in situ hybridization, we have examined alpha-MSH expression in the eye of the chicken during development. In the 1-day-old chick, alpha-MSH was expressed in the retinal pigment epithelial (RPE) cells, and also at a lower level in the cone cells. The melanocortin receptor subtypes, CMC1, CMC4 and CMC5, were expressed in the layers of the choroid and the neural retina, but not in the RPE cells. It is probable that the RPE cells secrete alpha-MSH to exert paracrine effects on the choroid and neural retina. During embryonic development, alpha-MSH immunoreactivity in the RPE cells was initially detected at embryonic day 10, and increased in intensity as development proceeded. No cone cells were stained with anti-alpha-MSH antiserum in any of the embryonic stages tested. The immunoreactivities for two prohormone convertases, PC1 and PC2, were co-localized to the RPE cells with a pattern of staining similar to that of alpha-MSH. Despite containing alpha-MSH immunoreactivity, the RPE cells in 1-day-old chicks expressed no immunoreactivity for the endoproteases. Furthermore, in a 3-day-old chick, pro-opiomelanocortin mRNA was detectable by in situ hybridization only in the photoreceptor layer and not in the RPE cells. These results suggest that the RPE cells and the cone cells are intraocular sources of alpha-MSH in the embryonic and postnatal life of the chicken respectively. Embryonic expression of alpha-MSH in the RPE cells implies a possible role for the peptide in ocular development.

By regulating cyclin-cyclin-dependent kinase (CDK) complex activity, individual CDK inhibitors (CDKIs) are potential tumor suppressors. One of the CDKIs, p27/Kip1, binds to a variety of CDK-cyclin complexes. A link between loss of p27/Kip1 function and development of pituitary tumors was suggested by the formation of pituitary tumors in almost all mice with germline deletion of the p27/Kip1 gene. However, genetic aberrations in the p27/Kip1 locus have not been analyzed in human pituitary tumors. We investigated eighteen non-functioning and GH-secreting pituitary tumor samples for p27/Kip1 mutations by single-strand conformational polymorphism (SSCP) following PCR. We found five abnormally migrating samples on the PCR-SSCP analysis. The sequence of these samples revealed a polymorphism of codon 109 (Val-->Gly), which has been previously described. No other structural changes of p27/Kip1 were found in these pituitary tumors within the coding region. In addition, no difference in p27/Kip1 protein levels in pituitary tumor tissues compared with normal pituitary tissues was demonstrated by immunostaining. These data suggest that both p27/Kip1 mutations and decreases in p27/Kip1 protein levels are infrequent in the development of pituitary tumors.

IGF-I has been implicated as a factor that may predispose one to prostate cancer and to benign prostatic hypertrophy (BPH). We established murine IGF-I transgenic mice under the control of rat probasin promoter and analysed the histology of the murine IGF-I-overexpressing prostate. Immunohistochemically, IGF-I was expressed in prostatic epithelial cells or basement membranes of the ventral, dorsal and lateral lobes in a line of IGF-I transgenic mice, but not in their control littermates. The anterior lobe did not express IGF-I. IGF-binding protein-3 (IGFBP-3), inhibitory to the mitogenic action of IGF-I, was detected in epithelial cells of prostatic ventral lobes, but not in those of the dorsal, lateral or anterior lobes of IGF-I transgenic mice. In controls, IGFBP-3 was not detected in epithelial cells of any prostatic lobe. Macroscopic prostatic size and the appearance of IGF-I transgenic mice were comparable with those of their control littermates of the same age. With a computed morphometric analysis, epithelial glands and intraglandular lumens in the prostatic lobes except the ventral lobe were smaller at 17 Months of age than at 14 Months both in IGF-I transgenic mice and controls. Glands and intraglandular lumens in the ventral prostatic lobes of IGF-I transgenic mice expressing more IGF-I protein in the prostate than controls were dense and enlarged similar to cysts compared with those of non-transgenic littermates without showing epithelial growth. Glands and lumens in the dorsal and lateral lobes of the IGF-I transgenic mice were also larger than controls at 14 and/or 17 Months of age. Glands in the anterior prostatic lobe of the IGF-I transgenic mice were not morphologically or morphometrically different from those of non-transgenic littermates. In conclusion, IGF-I transgenic mice under the control of rat probasin promoter showed more dense and enlarged epithelial glands in their prostatic ventral, dorsal and lateral lobes.