The effects of estrogen on prolactin gene methylation in normal and neoplastic rat pituitary tissues.

The effects of estrogen treatment on rat prolactin (PRL) gene methylation were analyzed in normal pituitaries and in three transplantable rat pituitary tumors. Northern analysis showed increased PRL mRNA expression in estrogen-treated pituitary and in MtT/F4 and MtT/F-DMBA tumors. Prolactin mRNA amounts in MtT/W15 tumor were decreased by estrogen treatment. There was an inverse relationship between changes in PRL mRNA expression and changes in gene methylation in the coding regions of the PRL gene after estrogen treatment. The amounts of the 4.6-Kb and 1.8-Kb restriction fragments generated by HpaII digestion in pituitary tissues were influenced by estrogen, with an increase in these fragments in normal pituitary, MtT/F4, and MtT/F-DMBA tumors after estrogen treatment. In contrast, the amounts of the 4.6-Kb and 1.8-Kb fragments were decreased in MtT/W15 tumors after estrogen treatment. Most of the internal -CCGG- sites in the PRL gene were methylated in the liver, and the PRL gene was not expressed in the liver. These data suggest that there is a tissue-specific pattern of DNA methylation of the PRL gene and that PRL gene methylation is influenced by estrogen in vivo in normal and tumorous pituitary tissues. These results also suggest that estrogen may influence PRL expression by multiple mechanisms, including changes in the level of DNA methylation.     

Detection of prolactin messenger RNA in rat anterior pituitary by in situ hybridization.

The effects of chronic diethylstilbestrol treatment on rat prolactin mRNA was analyzed by in situ hybridization histochemistry. Forty-day-old female rats were treated with 10 mg diethylstilbestrol in Silastic tubes for 3, 6, and 9 weeks. Estrogen treatment for 9 weeks increased pituitary wet weight (51.6 +/- 2.4 versus 7.9 +/- 0.31 mg for controls), serum prolactin (4155 +/- 571 versus 47.1 +/- 8.9 ng/ml for controls), and the percentage of immunoreactive prolactin cells (69% +/- 3% versus 34% +/- 2% for controls). In situ hybridization studies showed an increase in rat prolactin mRNA with increasing duration of estrogen treatment. After 9 weeks of estrogen treatment, there was a 2.3-fold increase in rat prolactin mRNA. 3H-cDNA was distributed diffusely throughout the anterior pituitary in both normal and hyperplastic pituitaries. There were no separate foci of adenomatous pituitary with increased labeling or with increased immunoreactive PRL cells. Although transplantable pituitary MtT/W15 tumors secreted very large amounts of PRL, compared with pituitaries from DES-treated rats, rat prolactin mRNA as evaluated by mean grain counts was considerably less in the MtT/W15 tumor than in DES-treated pituitary cells. These results show that in situ hybridization histochemistry can be used to detect changes in rat prolactin mRNA in tissue sections from the anterior pituitary with chronic estrogen treatment and that these pituitaries show a diffuse increase in immunoreactive prolactin cells and cellular prolactin mRNA, rather than distinct adenomatous areas within the glands.     

Diethylstilbestrol inhibits tumor growth and prolactin production in rat pituitary tumors.

Treatment of rats bearing transplantable MtT/W15 tumors with 10 mg of diethylstilbestrol (DES) for 3 weeks led to inhibition of tumor growth. The inhibition of tumor growth was reversible after removal of the DES. Histologic examination revealed decreased mitotic activity; however, DES did not produce cell necrosis. Concomitantly, the anterior pituitary glands of animals treated with DES became hyperplastic, with an increased number of prolactin (PRL)-producing cells. DES resulted in a decreased number of PRL cells in the tumor and decreased serum PRL/tumor weight, compared with that of control rats. There was also an increase in the number of growth hormone (GH) tumor cells and an increased serum GH/tumor weight. 17 beta-Estradiol had an effect similar to that of DES, while progesterone did not inhibit tumor growth or cause pituitary cell hyperplasia. Ovariectomy resulted in a decrease in the tumor growth rate, compared with that of control animals, suggesting that the MtT/W 15 tumors are relatively dependent on estrogens for optimal growth. These results indicate that DES inhibition of MtT/W 15 tumor growth is an excellent model for study of the mechanism of the inhibition of tumor growth and the modification of GH and PRL expression by the tumor cells.     

Analysis of S-100 protein positive folliculo-stellate cells in rat pituitary tissues.

The effects of diethylstilbestrol (DES) treatment and withdrawal on the folliculo-stellate (FS) cells in hyperplastic pituitaries were examined in Fischer 344 (F344) and Wistar Furth (W/F) rats by immunochemistry and electron microscopy. The presence of S-100 protein positive cells was examined by immunostaining in spontaneous and in transplantable rat pituitary tumors. The pituitaries of F344 rats showed a fivefold greater increase in weight in response to 5 weeks of DES treatment compared to pituitaries from W/F rats. S-100 protein (S-100) positive cells were significantly increased in both strains with hyperplastic pituitaries (P less than 0.05) 2 days after DES withdrawal. Ultrastructural studies revealed increased phagocytic activity in FS cells. S-100 positive cells were absent in both MtT/W15 and MtT/F4 transplantable tumors even after treatment with DES. Some spontaneous pituitary tumors in aged female rats had S-100 positive cells within the tumors at the periphery of the tumor nodules (3 of 12 cases), while most of these neoplasms did not contain S-100 positive cells. Incubation of normal dissociated pituitary cells with S-100 protein increased PRL secretion in vitro. The effectiveness of S-100 protein in increasing PRL secretion in vitro was less than thyrotropin releasing hormone (TRH). These results indicate that S-100 positive cells are present in normal and hyperplastic pituitaries, but not in spontaneous or in transplantable rat pituitary tumors and also suggest that the FS cells may exert a paracrine type regulation on PRL secretion.     

The effects of estrogens on tumor growth and on prolactin and growth hormone mRNA expression in rat pituitary tissues.

Estrogens inhibit tumor growth and modify PRL and GH expression in the MtT/W15 transplantable rat pituitary tumor. The effects of estradiol (E2) and diethylstilbestrol (DES) on PRL and GH mRNA levels were investigated. Estrogens increased GH mRNA levels and decreased PRL mRNA levels as detected by in situ hybridization and Northern blot hybridization with oligonucleotide probes, while inhibiting tumor growth. Similar changes in immunoreactive GH and PRL were seen in the tumor cells. The pituitary glands of tumor-bearing rats treated with estrogen for 3 weeks were increased in weight with a concurrent increase in pituitary PRL mRNA when analyzed by dot blot hybridization. These results indicate that estrogens have an inhibitory effect on the growth of the MtT/W15 tumor and increase GH protein and mRNA levels, while causing PRL protein and mRNA levels to decrease. The pituitaries of tumor-bearing rats concomitantly undergo PRL cell hyperplasia with an increase in PRL mRNA. These results also demonstrate a paradoxical effect of estrogens on different pituitary tissues.     

Regulation of estrogen receptor mRNA in normal and neoplastic rat pituitary tissues

The effects of estradiol 17β (E₂) on the regulation of estrogen receptor (ER) mRNA amounts in normal and neoplastic rat pituitary tissues were analyzed by in situ hybridization with an oligonucleotide probe. E₂ treatment produced a significant increase in ER mRNA amounts in two transplantable pituitary tumors (MtT/Wl5 and MtT/F4) and in the GH₃ cell line. ER mRNA amounts were also increased In normal pituitaries after 6 weeks of E₂ treatment, but the differences were not significant. Biochemical assay of ER proteins confirmed the presence of ER protein in MtT/Wl 5 and MtT/F4 tumors. “Cytoplasmic” ER proteins were decreased by E₂ in the MtT/Wl5 tumor.These results indicate that ER mRNA is present in normal pituitaries and in pituitary tumors and can be regulated by estrogen treatment in vivo and in vitro. The inhibitory effects of high estrogen concentration on proliferation of transplantable pituitary tumors in vivo and GH₃ cells in vitro is not explained by the absence of ER mRNAs in these tumors.     

Analysis of prolactin and growth hormone production in the MtT/F4 transplantable pituitary tumor by the reverse hemolytic plaque assay.

The reverse hemolytic plaque assay (RHPA) was used to detect hormone secretion from normal pituitary cells and from the transplantable MtT/F4 pituitary tumor cells. Aliquots of the same cell suspensions were analyzed by immunocytochemistry (ICC). Normal pituitaries had more growth hormone (GH)-producing cells than tumors when analyzed by both the RHPA and ICC. However, the MtT/F4 tumor had significantly more prolactin (PRL)-secreting cells. Mammosomatotropic (MS) cells, which produced both PRL and GH, were identified in both normal and tumorous pituitaries with the RHPA and ICC. A combined procedure of RHPA followed by ICC staining on the same slide also revealed MS cells in both normal and tumorous pituitary cells, although the percentage of MS with this technique was less than with the other two methods. These results show that MS cells from a significant population of cells in the MtT/F4 tumor and that the RHPA and ICC can be used to study the regulation of this cell type.     

Effects of estrogens on pituitary cell and pituitary tumor growth

Estrogens have been known to induce PRL cell hyperplasia in the anterior pituitary of some species for many decades. Recent studies have shown variable susceptibility to estrogen-induced hyperplasia in different strains of rats. The distinction between hyperplastic pituitaries and adenomas is usually not made by most investigators in this field, although true neoplasms can usually be propagated by serial transplantation. The growth of transplantable tumors is usually inhibited by estrogen in vivo. Estrogens have a biphasic effect on pituitary cell proliferation in vitro with higher concentrations of estradiol inhibit cell growth, and lower concentrations stimulating PRL secretion. Estrogens can regulate PRL gene methylation in vivo thus affecting PRL mRNA expression. Recent studies have suggested that estrogen regulates signal transduction by stimulating protein kinase C. Estrogens also regulate specific proto-oncogenes such as c-myc and c-fos. These observations may help to explain some of the regulatory effects of estrogens on cell proliferation and tumor development.     

Interactions between transplanted pituitary tumor and gonads in nude mice

More pronounced hyperprolactinemia occurred in the male nude mice bearing a transplantable rat pituitary tumor than in the female nude mice, although the hyperprolactinemia in rats bearing the identical tumors was more prominent in the females than in the males. PRL levels in castrated nude mice of both sexes bearing tumor were almost the same as those in the intact female nude mice. PRL levels in the tumor tissues in each group paralleled relative abundance of PRL-secreting cells in the tumors. Supplement with testosterone or estrogen to the castrated mice recovered the PRL concentrations to the same or higher levels than those in intact male mice. Reproductive organs in the tumor-bearing male nude mice showed no alterations, whereas those in the female nude mice were markedly atrophic. Tumors had receptors for estrogen but not for androgen. Insufficient estrogen secretion due to the ovarian atrophy caused by the PRL from the tumors seemed responsible for the failure in growth of PRL-secreting tumor cells and consequent low PRL production by the tumor.     

Morphologic effects of bromocriptine on spontaneously occurring pituitary prolactin-cell hyperplasia in old Long-Evans rats.

The effect of bromocriptine (BEC), a dopaminergic agonist, on nontumorous pituitary prolactin (PRL) cells of aging female Long-Evans rats, was studied histologically, immunocytologically, electron-microscopically, and morphometrically. Rats were arbitrarily divided into two control groups, one with normal (less than 20 ng/ml) and one with elevated serum PRL concentrations, and into four BEC-treated groups, all of which had increased serum PRL levels prior to commencement of BEC administration. In hyperprolactinemic control rats, compared with normoprolactinemic control rats, pituitary weight and percentage of pituitary PRL cells were increased. The morphologic features of PRL cells in these two groups did not differ markedly, which suggested that hyperprolactinemia was due to increased PRL-cell number and not increased PRL-cell function. Compared with age-matched hyperprolactinemic control rats, hyperprolactinemic rats treated with BEC showed a reversible decrease in serum PRL levels, pituitary weight as well as percentage of pituitary PRL cells, and by ultrastructural morphometry an increase in the volume density of lysosomes. BEC caused no striking changes in nuclear and cytoplasmic areas, volume densities of RER, Golgi regions, mitochondria, lipid droplets, and size and volume densities of forming and storage granules. Since spontaneously hyperplastic PRL cells show less conspicuous morphologic changes following BEC treatment than PRL cells rendered hyperplastic by estrogen administration or pituitary transplantation, it is suggested that PRL cells with no increased endocrine function respond less markedly to dopaminergic suppression than endocrinologically hyperactive PRL cells. It can be concluded that BEC suppresses spontaneous proliferation of PRL cells which occurs with aging.     

Estrogen-induced hyperplasia and neoplasia in the rat anterior pituitary gland. An immunohistochemical study

Diethylstilbestrol (DES) treatment of weanling F344 female rats resulted in enlarged pituitary glands and diffuse pituitary prolactin (PRL) cell hyperplasia in all animals after 9 and 12 weeks of treatment. Serum PRL was significantly greater than in control rats (P less than 0.001). Immunohistochemical studies showed that most of the pituitary gland cells consisted of PRL cells. Ultrastructural studies showed increased numbers of PRL cells with hyperplasia of the rough endoplasmic reticulum and decreased numbers of secretory granules. There was a decrease in the relative number of growth hormone (GH) and other cell types in the anterior pituitary. Pituitary tumors and normal pituitary glands were dissociated with trypsin and maintained in culture for 3 weeks. The numbers of PRL and GH cells decreased with time in both groups, and there was an increase in the number of fibroblasts. Staining of the culture cells with neuron-specific enolase showed that the anterior pituitary cells were positive for this enzyme, while the fibroblastic cells were negative. When dissociated pituitary cells were cultured in the presence of 10(-9) M DES for 7 days, there was a 42% increase in the number of immunoreactive PRL cells. These results indicate that DES-treated rats provide an excellent model for study of the in vivo and in vitro regulation of pituitary hyperplasia and neoplasia.     

FINE STRUCTURAL STUDIES OF RAT ADENOHYPOPHYSIS —EFFECTS OF EXOGENOUS GROWTH HORMONE AND HYPOTHALAMIC LESIONS ON SOMATOTROPHS

The response of rat pituitary somatotrophs to transplantation of the MtT-W10 pituitary tumor and prolonged treatment with large doses of purified bovine growth hormone was examined. The effects of hypo-thalamic lesions on the somatotrophs were also studied.
Involution occurred in somatotrophs of the anterior pituitary of rats bearing a transplantable tumor. The changes in the somatotrophs were observable at 4 weeks and consisted of smaller cells and fewer secretory granules.
Administration of growth hormone produced a triphasic response; (1) increased cross-sectional area of cell, increased size and number of secretory granule, (2) return of parameters to normal levels, and (3) suppression of somatotrophs by exogenous growth hormone.
Following destruction of ventromedial nucleus (VMN), the number of secretory granules per somatotroph increased. Concomitantly, there was an enlargement of the mean diameter as well as an increase in the mean surface area of the somatotrophs. After destruction of dorsomedid nucleus (DMN), the number of secretory granules decreased rapidly and reached the lowest level. Two days after the operation, the secretory granules re-accumulated very slowly in the cytoplasm but remained below control level. Accompanying the decreased granule count, the mean diameter of the largest granule was smaller than that of the controls during all experimental periods.     

Pituitary Folliculo-Stellate-Like Cells Stimulate Somatotroic Pituitary Tumor Growth in Nude Mice

A new cell line (TtT/GF) established from a murine pituitary thyrotropic tumor having characteristics similar to those of pituitary folliculo-stellate cell (FS cell) was implanted into nude mice together with cells from a rat pituitary somatotrophic tumor cell line (MtT/S) to determine whether the former enhances pituitary tumor growth. For as long as 2-3 mo after implantation, MtT/S cells implanted either alone or together with fibroblasts formed either no tumors or only very small tumors in the nude mice. In contrast all of the nude mice that had received MtT/S cells implanted together with TtT/GF cells developed large tumors. Furthermore, the mice bearing the MtT/S and TtT/GF implants showed a significantly higher body weight and serum growth hormone level than those bearing only MtT/S cells or a combination of MtT/S cells and fibroblasts. The TtT/GF cell line itself had no tumorigenicity during the experimental period. Therefore, the TtT/GF cell line as a model of FS cells enhanced pituitary endocrine cell tumor formation. Additionally, immunocytochemistry showed that TtT/GF cells positive for glial fibrillary acidic protein (GFAP) or S-100 protein were present in the parenchymatous tissue elements or connective tissue surrounding the tumor nests. In the parenchymatous tissue, the TtT/GF cells exhibited a stellate appearance and surrounded neighboring tumor cells with their long cell processes. These results suggest that TtT/GF cells can serve as a model for pituitary FS cells, and are capable of stimulating pituitary tumor growth either by modifying the microenvironment or producing growth factors.     

Biological aspects of pituitary tumors induced by synthetic salmon calcitonin (TZ-CT) in Sprague-Dawley rats. Immunohistochemical and ultrastructural studies.

Rat pituitary tumors induced by synthetic salmon calcitonin (TZ-CT) were studied by the indirect peroxidase-labeled antibody method, together with ultrastructure and serum hormone measurement. Immunohistochemically, TZ-CT-induced pituitary tumors showed staining for only rLH alpha subunit, and were negative for other peptide hormones including GH, PRL, alpha MSH and ACTH, and the beta subunit of glycoprotein hormones. Electron microscopic examination showed that the majority of tumor cells possessed numerous small secretory granules, 100 to 200 nm in diameter. The serum PRL concentrations of rats with TZ-CT-induced pituitary tumors were markedly elevated, but not beyond 130 ng/ml. From our data, TZ-CT-induced pituitary tumors are considered to be endocrinologically inactive and to produce alpha subunit. Furthermore, these tumors are thought to be potentially useful models of alpha subunit-producing pituitary tumors in humans. This is the first report to document the tumorigenesis of alpha subunit-producing pituitary tumors in rats after long-term treatment with calcitonin.     

Biological Aspects of Pituitary Tumors Induced by Synthetic Salmon Calcitonin (TZ–CT) in Sprague-Dawley Rats

Rat pituitary tumors induced by synthetic salmon calcitonin (TZ–CT) were studied by the indirect peroxidase-labeled antibody method, together with ultrastructure and serum hormone measurement. Immunohistochemically, TZ–CT-induced pituitary tumors showed staining for only rLHα subunit, and were negative for other peptide hormones including GH, PRL, αMSH and ACTH, and the α subunit of glycoprotein hormones. Electron microscopic examination showed that the majority of tumor cells possessed numerous small secretory granules, 100 to 200 nm in diameter. The serum PRL concentrations of rats with TZ–CT induced pituitary tumors were markedly elevated, but not beyond 130 ng/ml. From our data, TZ–CT-induced pituitary tumors are considered to be endocrinologically inactive and to produce α subunit. Furthermore, these tumors are thought to be potentially useful models of α subunit producing pituitary tumors in humans. This is the first report to document the tumorigenesis of α subunit producing pituitary tumors in rats after long-term treatment with calcitonin.     

Galectin-3 expression in functioning and silent ACTH-Producing adenomas

Galectin-3 (Gal-3), a β galactoside-binding protein, has been implicated in a variety of biological functions including cell growth, differentiation, tumor cell adhesion, angiogenesis, tumor progression, and metastasis. We recently reported that Gal-3 was expressed in a subset of normal pituitary cells and tumors including PRL, ACTH, and in folliculostellate (FS) cells and tumors [1,2] and that Gal-3 had an important regulatory role in pituitary cell proliferation [1]. We further investigated the expression of Gal-3 protein in ACTH- and PRL-producing tumors and the expression of various galectin mRNAs by RT-PCR in pituitary adenomas and normal pituitary. Most silent ACTH subtypes 1 and 2 adenomas were negative or only focally positive for Gal-3 expression compared to functioning ACTH tumors from patients with Cushing’s disease and Nelson’s syndrome. In the normal pituitary, Gal-3 was expressed in less than 1% of the basophil-invading cells (ACTH cells present in the posterior pituitary) and in a subset of the anterior lobe ACTH-positive cells. RT-PCR analyses showed that many members of the galectin family including galectins 1, 2, 3, 4, 5, 6, 7, 8, and 9 were expressed in normal pituitary and in functioning ACTH- and PRL-producing tumors. These results indicate that Gal-3 is associated with functioning ACTH and PRL tumors and is expressed infrequently in silent ACTH adenomas, suggesting that Gal-3 protein and/or gene is altered in non-functioning ACTH tumors. The use of ACTH and Gal-3 immunostaining should help in the diagnosis of silent ACTH adenomas.     

The Expression of Wnt4 Is Regulated by Estrogen via an Estrogen Receptor Alpha-dependent Pathway in Rat Pituitary Growth Hormone-producing Cells

Wnt signaling is important in many aspects of cell biology and development. In the mouse female reproductive tract, Wnt4, Wnt5a, and Wnt7a show differential expression during the estrus cycle, suggesting that they participate in female reproductive physiology. Although the pituitary is a major gland regulating reproduction, the molecular mechanism of Wnt signaling here is unclear. We elucidated the subcellular distribution of Wnt4 in the pituitary of estrogen-treated ovariectomized female rats. Expression of Wnt4 mRNA increased dramatically, particularly in proestrus compared with estrus and metestrus. Wnt4 protein was observed in the cytoplasm of almost all growth hormone (GH)-producing cells and in only a few thyroid-stimulating hormone β (TSHβ)-producing cells. In rat GH-producing pituitary tumor (MtT/S) cells, estrogen-induced expression of Wnt4 mRNA was completely inhibited by estrogen receptor antagonist ICI 182,780 in vitro. Thus, rat pituitary GH cells synthesize Wnt4 and this is induced by estrogen mediated via an estrogen receptor alpha-dependent pathway.