ACTH-positive Cells Research Articles

Evaluation of Prenatal Transportation Stress on the Number of Pituitary Corticotrophs in Mature Brahman Cows

Abstract Prenatal transportation stress (PNS) results in calves that are more temperamental and have greater circulating concentrations of cortisol compared to control calves. The objective of this experiment was to evaluate whether PNS alters the number of pituitary corticotrophs in mature Brahman cows. We hypothesized that the increased circulating cortisol concentrations previously characterized in this bovine model is associated with developmental changes in the anterior pituitary leading to an increased number of corticotrophs. Pregnant Brahman cows (n = 48) were transported in trailers for 2-hour periods at 60±5, 80±5, 100±5, 120±5, and 140±5 days of gestation. Non-transported pregnant cows (n = 48) were designated as the Control group. Control and PNS offspring heifers were managed together under the same environmental conditions. At approximately 5 yr of age, randomly selected non-pregnant cows (Control, n = 8; PNS, n = 6) were humanely harvested and the whole pituitaries were collected. Pituitaries were weighed, fixed in paraformaldehyde, serially dehydrated with graded ethanol, embedded in paraffin blocks, and cut into 5-μm sections. Immunohistochemistry was performed to detect cells expressing adrenocorticotropic hormone (ACTH) as a marker for corticotrophs. Three comparable sections from the midsagittal plane from each animal were processed using an ovine ACTH-specific antibody (Dr. A.F. Parlow, NIDDK). Five fields of view were analyzed per section (15 fields per animal). Anterior pituitary gland weight did not differ (P > 0.10) between groups (Control = 2.11 ± 0.12 g; PNS = 2.10 ± 0.15 g). The mean number of ACTH-positive cells between control (531±56 cells/section) and PNS cows (477±49 cells/section) also did not differ (P > 0.10). In conclusion, the number of pituitary corticotrophs in mature Brahman cows was not affected by prenatal transportation stress and is likely not a mechanism mediating the increased circulating cortisol concentrations seen in this bovine model of fetal programming.

Human anterior pituitary's ACTH cells during the aging process: immunohistochemic and morphometric study.

Corticotrophs produce a hormone that stimulates the adrenal gland cortex to secrete glucocorticoids, which in turn have effects on carbohydrate and protein metabolism. Quantification, morphological characteristics, and distribution of corticotrophs in the anterior pituitary and changes in the number and shape of the cells during aging have been examined using immunohistochemical and morphometric methods. The material consisted of 14 anterior pituitaries taken from cadavers at routine autopsy. The tissue was processed by standard histological procedure and the obtained slices were stained by the monoclonal anti-ACTH antibody for corticotrophs identification. Digital images of stained histological sections were analyzed using the morphometric method with the Image J system. The volume density of ACTH positive cells was determined. The cases were classified into three age groups. One-way ANOVA showed that the volume density of the corticotrophs was significantly higher in the second and third group in relation to the first group. The difference in the volume densities of the corticotrophs between the genders was not significant. Morphometric and statistical analyses demonstrated a significant positive correlation between the corticotrophs volume densities and the age of the evaluated cases. Linear regression showed that age significantly predicts corticotrophs volume density. Corticotrophs significantly increase during the life span.

MON-469 Pituitary Corticotroph Lesions: Clinical, Biochemical, and Pathological Characterization of Cushing Disease, Silent Corticotroph Adenomas, and Corticotroph Hyperplasia

Pituitary adenomas with positive ACTH staining are derived from the corticotroph lineage. Most corticotroph adenomas are small and secrete ACTH, causing a form of hypercortisolism known as Cushing disease (CD); however, a subset do not secrete ACTH and are referred to as silent corticotroph adenomas (SCA). Rarely, hypercortisolemic patients who undergo surgery are diagnosed with the ill-defined entity of corticotroph hyperplasia (CH). In order to better define CH and compare the clinical, laboratory, and imaging parameters of CD, SCA, and CH, comprehensive chart review was performed for all patients at our institution within the past ten years who underwent transsphenoidal surgery for a pituitary mass showing diffuse ACTH immunostaining in lesional tissue. All cases were re-reviewed by an experienced neuropathologist to confirm the diagnoses. Patients with clinical and biochemical evidence of ACTH-dependent hypercortisolemia and an ACTH-positive adenoma were classified as CD (n=59). Patients without hypercortisolemia but with ACTH-staining adenomas were classified as SCA (n=32). Patients clinically suspected of having Cushing disease whose surgical specimen did not show adenoma but consisted only of abnormally expanded acini with ACTH-positive cells were classified as CH (n=13). Average (± SD) pre-operative cortisol (mcg/dL)/ ACTH (pg/mL) measurements were: CD 23.7±8.1 / 66.4±40.1; SCA 12.7±6.2 / 44.4±38.0; CH 24.5±5.9 / 63.8±35.1. SCA patients had larger tumors and presented with more symptoms of compression whereas both CD and CH patients presented with characteristic features of hypercortisolism: weight gain, hypertension, hirsutism, and skin changes (p<0.05 compared to SCA). Compared to CD patients, CH patients more frequently exhibited mood changes (p=0.007) and cognitive dysfunction (p=0.002). CH patients without prior pituitary surgery on average had a greater absolute number of negative late-night salivary cortisol results than CD patients (p=0.006). Of the patients with adequate follow-up, CH patients had higher cortisol levels 6 months post-operatively (p=0.007). Most patients with CH (8/13, 61.5%) had discrete lesions identified on pre-operative MRI. CD patients were more likely to have a higher MIB-1 proliferative index than those of CH patients (CD: 2.8±3.7, CH: 0.4±0.2, p=0.029). In conclusion, SCAs are more likely to cause symptoms of compression, while CD and CH cause symptoms related to hypercortisolism. There may be subtle differences between CD and CH patients, but both groups manifest with ACTH-dependent hypercortisolism and can be nearly indistinguishable pre-operatively. The difference in MIB-1 indices suggests that CH may represent a reactive or pre-neoplastic condition as opposed to undersampled adenoma. Further studies are needed to better understand the pathophysiology of corticotroph cell hyperplasia and its relation to CD.

Erratum. Value of pituitary gland MRI at 7 T in Cushing's disease and relationship to inferior petrosal sinus sampling: case report.

Cushing's disease is caused by adrenocorticotrophic hormone (ACTH)-secreting pituitary adenomas, which are often difficult to identify on standard 1.5-T or 3-T MRI, including dynamic contrast imaging. Inferior petrosal and cavernous sinus sampling remains the gold standard for MRI-negative Cushing's disease.The authors report on a 27-year-old woman with Cushing's disease in whom the results of standard 1.5-T and 3-T MRI, including 1.5-T dynamic contrast imaging, were negative. Inferior petrosal sinus sampling showed a high central-to-peripheral ACTH ratio (148:1) as well as a right-to-left ACTH gradient (19:1), suggesting a right-sided pituitary microadenoma. The patient underwent 7-T MRI, which showed evidence of a right-sided pituitary lesion with focal hypoenhancement not visualized on 1.5-T or 3-T MRI. The patient underwent an endoscopic endonasal transsphenoidal operation, with resection of a right-sided pituitary mass. Postoperatively, she developed clinical symptoms suggestive of adrenal insufficiency and a nadir cortisol level of 1.6 μg/dl on postoperative day 3, and hydrocortisone therapy was initiated. Permanent histopathology specimens showed Crooke's hyaline change and ACTH-positive cells suggestive of an adenoma.MRI at 7 T may be beneficial in identifying pituitary microadenoma location in cases of standard 1.5-T and 3-T MRI-negative Cushing's disease. In the future, 7-T MRI may preempt inferior petrosal sinus sampling and help in cases of standard and dynamic contrast 1.5-T and 3-T MRI-negative Cushing's disease.

Multihormonal pituitary adenoma concomitant with Pit-1 and Tpit lineage cells causing acromegaly associated with subclinical Cushing\u2019s disease: a case report

BackgroundA functional pituitary adenoma can produce multiple anterior-pituitary hormones, such as growth hormone (GH) -producing adenomas (GHoma) with prolactin or thyrotropin stimulating hormone production in the same lineage. However, it is very rare that acromegaly shows subclinical Cushing’s disease (SCD) beyond the lineage. Here we describe the involvement of intratumoral coexistence with 2 types of hormone-producing cells associated with different lineage in acromegaly concomitant with SCD.Case presentationIn our study, we performed clinical evaluation of the patient showing acromegaly with SCD. To elucidate the mechanisms of this pathology, we analyzed immunohistochemistry and gene expression of anterior-pituitary hormones and transcriptional factors in the resected pituitary tumor. On immunohistochemical staining, most of the tumor cells were strongly stained for GH antibody, while some cells were strongly positive for adrenocorticotropic hormone (ACTH). Gene expression analysis of a transsphenoidal surgery sample of the pituitary gland revealed that ACTH-related genes, such as POMC, Tpit, and NeuroD1 mRNA, had higher expression in the tumor tissue than the nonfunctional adenoma but lower expression compared to an adenoma of typical Cushing’s disease. Further, double-labeling detection methods with a fluorescent stain for ACTH and GH demonstrated the coexistence of ACTH-positive cells (GH-negative) among the GH-positive cells in the tumor. Additionally, Pit-1 expression was reduced in the ACTH-positive cells from tumor tissue primary culture.ConclusionHere we described a case of a pituitary tumor diagnosed with acromegaly associated with SCD. We performed quantitative-expression analyses of transcriptional factors of the tumor tissue and immunohistochemistry analysis of tumor-derived primary culture cells, which suggested that the multihormonal pituitary adenoma concomitant with Pit-1 and Tpit lineage cells caused acromegaly associated with SCD.

Endothelial immunocytochemical expression of pituitary IL-1β and its relation to ACTH-positive cells is regulated by corticosterone in the male rat

Interleukin-1 beta (IL-1β) is a cytokine linking the neuroendocrine system and metabolic homeostasis. We have previously demonstrated the relevance of IL-1β for maintaining the pituitary ACTH-producing cells by immuno-blocking its effects in pituitary cultures. However, the morphological characteristics and the intimate relationship of the pituitary cells expressing IL-1β and ACTH remain unknown. For determining pituitary variations of immunoreactivity for IL-1β and its relation with ACTH-positive cells under stress situations, we performed an immunohistochemical analysis of the expression of IL-1β and ACTH in the pituitary gland of adult rats, in the absence or presence of corticosterone, by establishing different groups: untreated, sham-operated, and bilaterally adrenalectomized animals. In the rats subjected to surgery, the glucocorticoid was administered on the same day of the intervention and on the third day post-surgery. Interestingly, it was observed that IL-1β was located in the pituitary endothelial cells at the hypophyseal portal vessels, regardless of the treatment schedule. When comparing the pituitary immunoreactive surface to IL-1β expression without corticosterone, adrenalectomized animals displayed a significantly greater area than the sham-operated animals. Corticosterone significantly inhibited the effect of adrenalectomy depending on the time interval it was administered. By in situ hybridization, IL-1β mRNA expression was also correlated with immnunocytochemical expression of pituitary IL-1β. Our results demonstrate that IL-1β is a constitutive element in endothelial portal pituitary vessels and under stress experimental conditions IL-1β increases its expression and its relation with ACTH-positive cells, suggesting that IL-1β could participate in an autocrine-paracrine fashion thereby modulating the pituitary population of ACTH-positive cells.

Effects of Carbenoxolone on the Canine Pituitary-Adrenal Axis.

Cushing’s disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to suppressive negative feedback by glucocorticoids. The abnormal expression of 11beta-hydroxysteroid dehydrogenase (11HSD), which is a cortisol metabolic enzyme, is found in human and murine corticotroph adenomas. Our recent studies demonstrated that canine corticotroph adenomas also have abnormal expression of 11HSD. 11HSD has two isoforms in dogs, 11HSD type1 (HSD11B1), which converts cortisone into active cortisol, and 11HSD type2 (HSD11B2), which converts cortisol into inactive cortisone. It has been suggested that glucocorticoid resistance in corticotroph tumors is related to the overexpression of HSD11B2. Therefore it was our aim to investigate the effects of carbenoxolone (CBX), an 11HSD inhibitor, on the healthy dog’s pituitary-adrenal axis. Dogs were administered 50 mg/kg of CBX twice each day for 15 days. During CBX administration, no adverse effects were observed in any dogs. The plasma adrenocorticotropic hormone (ACTH), and serum cortisol and cortisone concentrations were significantly lower at day 7 and 15 following corticotropin releasing hormone stimulation. After completion of CBX administration, the HSD11B1 mRNA expression was higher, and HSD11B2 mRNA expression was significantly lower in the pituitaries. Moreover, proopiomelanocortin mRNA expression was lower, and the ratio of ACTH-positive cells in the anterior pituitary was also significantly lower after CBX treatment. In adrenal glands treated with CBX, HSD11B1 and HSD11B2 mRNA expression were both lower compared to normal canine adrenal glands. The results of this study suggested that CBX inhibits ACTH secretion from pituitary due to altered 11HSD expressions, and is potentially useful for the treatment of canine Cushing’s disease.

VE1 antibody immunoreactivity in normal anterior pituitary and adrenal cortex without detectable BRAF V600E mutations.

The VE1 monoclonal antibody was developed to recognize the V600E mutation in BRAF, which is found in various tumors. We report that the VE1 antibody stains normal anterior pituitary gland and adrenal cortex, which lack detectable BRAF V600E mutations. Staining with the VE1 antibody was seen in the adenohypophysis and correlated well with adrenocorticotropic hormone (ACTH)-positive cells. ACTH-positive cells were typically most concentrated in the central mucoid wedge and pars intermedia, and VE1 staining was strong in these regions. Moreover, VE1 staining was seen in ACTH-expressing pituitary adenomas without detectable BRAF mutations. VE1 staining of the adrenal cortex was also significant, with the strongest staining seen in the inner segment of the zona fasciculata. Parathyroid glands, pancreatic islets, or parafollicular C cells in the thyroid showed no VE1 staining. Overall, VE1 staining of endocrine tissues strongly suggests limitations on the use of this antibody for the detection of BRAF mutations.

AGE-RELATED CHANGES OF THE PITUITARY FOLLICULO-STELLATE CELLS IN RATS IN CHRONIC STRESS

Folliculo-stellate cells are known as S-100 protein immunopositive cells of the anterior lobe of pituitary gland which are not secreting hormones and are presumed to be organ specific stem cells of the adenohypophys. Their role in adaptation of the body to stress remains unclear. To evaluate dynamics of folliculo-stellate and hormone-producing cells in rats of different age in chronic stress exposure. Sprague-Dawley rats aged 1, 3, 6 and 12 months were exposed to chronic immersion immobilization stress. Histological section of the pituitary glands were stained immunohistochemically with subsequent image analysis. In control rats S-100 protein-immunopositive cells increased in number with age and negatively correlated with the number of ACTH-positive cells. In experimental animals aged 1 and 3 months volume density of S-100 protein-immunoreactive cells significantly decreased and negatively correlated with ACTH-positive cells, while in 6 month old rats it was only slightly decreased and in 12 months old animals showed tendency to increase. Results of the study demonstrating capacity of the folliculo-stellate cells to contribute to the plasticity of the hypothalamo-hypophyseo-adrenal axis in chronic stress exposure at different age.

The impact of water-floating and high-intensity exercise on rat’s HPA axis and interleukins concentrations

Our studies explore the changes of blood corticosterone (CORT), adrenocorticotropic hormone (ACTH), interleukin (IL)-1β, IL-2, IL-6 concentrations and the pituitary ACTH expression in rats after water floating in the presence or absence of following high-intensity exercise. The rats were randomly assigned into three groups. Group A served as control; Group B received 180 minutes water floating and psychological (fear) stimulation; Group C received the same treatment as Group B in addition and 120-minutes non-stop running. Compared to Group A, Group B showed a significant increase of IL-2 (19.91 ± 2.52 vs. 13.09 ± 3.13 ng/ml, P < 0.05), and IL-6 (0.18 ± 0.08 vs. 0.12 ± 0.05 ng/ml, P < 0.05); Group C demonstrated a significant increase of CORT (977.22 ± 207.36 ng/ml vs. 434.58 ± 110.45 ng/ml, P <0.01) and IL-1β (0.21 ± 0.04 vs. 0.16 ± 0.06 ng/ml, P < 0.05), IL-2 (20.29 ± 4.23 vs. 13.09 ± 3.13 ng/ml, P < 0.05), and IL-6 (0.19 ± 0.03 vs. 0.12 ± 0.05 ng/ml, P < 0.05) levels, and a significant decrease of ACTH (16.95 ± 5.46 vs. 22.96 ± 7.32 pg/ml, P = 0.03). Immunohistochemical staining showed the decreased number of pituitary ACTH-positive cells in both Groups B and C (P < 0.05) as compared to Group A. These results have lead us to believe that acute psychological stress can activate the pituitary-adrenal axis and lead to elevation of serum IL-2, IL-6 concentrations. Combined with high-intensity exercise, it can result in the increase of serum CORT, IL-1β, IL-2, IL-6 levels, and the suppression of ACTH.

Expression of genes related to corticotropin production and glucocorticoid feedback in corticotroph adenomas of dogs with Cushing’s disease

Cushing’s disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to negative feedback by glucocorticoids. In this study, we examined gene expression related to adrenocorticotropic hormone (ACTH) production and secretion, and the negative feedback by glucocorticoids in canine corticotroph adenoma. We used resected corticotroph adenomas from 10 dogs with Cushing’s disease. In order to investigate the alteration of gene expression between corticotroph adenoma and normal corticotrophic cells, ACTH-positive cells in the anterior lobe were microdissected using a laser-capture microdissection system, and mRNA levels of proopiomelanocortin (POMC), corticotropin releasing hormone receptor 1 (CRHR1), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11 beta hydroxysteroid dehydrogenase (11HSD) type 1 and type 2 were determined using real-time RT-PCR. POMC, CRHR1, and 11HSD2 mRNA levels in corticotroph adenoma were greater than those in normal corticotrophic cells (POMC, 5.5-fold; CRHR1, 4.9-fold; 11HSD2, 4.2-fold, P < 0.01, respectively). MR and 11HSD1 mRNA levels in corticotroph adenoma were lower than those in normal corticotrophic cells (MR, 2.2-fold; 11HSD1, 2.9-fold, P < 0.01, respectively). GR mRNA levels did not differ between corticotroph adenoma and normal corticotrophic cells. Our results may help to understand the increased ACTH production and the resistance to negative feedback suppression by glucocorticoids in canine corticotroph adenomas. These changes in gene expression may have a role in the growth of canine corticotroph adenoma, and help elucidate the pathophysiology of dogs with Cushing’s disease.

Vasopressin Mediates Mitogenic Responses to Adrenalectomy in the Rat Anterior Pituitary

To determine whether increased vasopressinergic activity during chronic stress or adrenalectomy mediates trophic changes in the corticotroph, we examined the effect of peripheral V1 receptor blockade in rats, using the antagonist, dGly[Phaa1,D-tyr(et), Lys, Arg]vasopressin (VP), on the number of pituitary cells taking up bromodeoxyuridine (BrdU) and cells containing immunoreactive ACTH (irACTH). Adrenalectomy significantly increased the number of BrdU- and ACTH-labeled cells at 3 and 6 d, and a much larger increase was observed at 28 d. Minipump infusion of V1 antagonist for 28 d, at doses blocking the increases in ACTH and corticosterone induced by exogenous VP, prevented the increases in BrdU incorporation, but not irACTH cells observed 28 d after adrenalectomy. Unexpectedly, colocalization of BrdU with ACTH-positive cells was minor (about three cells per pituitary section), and this was unaffected by adrenalectomy or V1 antagonist infusion. In contrast, adrenalectomy for 6 or 14 d failed to increase BrdU incorporation or irACTH cells in V1b receptor knockout mice while inducing the expected increase in wild-type mice. The data show that VP is required for pituitary mitogenesis after adrenalectomy but, at least in rats, not for increasing the number of corticotrophs. The lack of colocalization of ACTH in mitotic cells suggests that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells.

Octreotide and the Novel Multireceptor Ligand Somatostatin Receptor Agonist Pasireotide (SOM230) Block the Adrenalectomy-Induced Increase in Mitotic Activity in Male Rat Anterior Pituitary

The novel somatostatin receptor agonist pasireotide binds with high affinity to somatostatin receptors SSTR1, 2, 3, and 5. Acting principally through the latter, it inhibits basal and CRH-stimulated ACTH secretion from the AtT20 corticotroph cell line and ACTH release from a proportion of human corticotroph adenomas both in vitro and in vivo. Data supporting an additional antiproliferative effect has led to pasireotide being explored as a potential therapy for patients with Cushing's disease. We have compared the effects of pasireotide and octreotide on adrenalectomy-induced mitotic and apoptotic activity in the male rat anterior pituitary. Adrenalectomized rats were treated with daily sc injections of vehicle, pasireotide, or octreotide. Changes in proliferation and apoptosis were determined 2-6 d postoperatively. Pasireotide and octreotide had no effect on baseline pituitary cell turnover and no measurable effects on apoptosis. However, the wave of increased mitotic activity normally seen in the pituitary after adrenalectomy was completely abolished. Nevertheless, pasireotide and octreotide did not diminish the increase in ACTH-immunopositive cell index after adrenalectomy, indicating that cell division and differentiation of hormonally null cells in the pituitary are under independent control. In conclusion, basal cell turnover in the pituitary is not inhibited by pasireotide or octreotide. Bilateral adrenalectomy stimulates differentiation of preexisting null cells into ACTH-positive cells. Cell division after bilateral adrenalectomy occurs in a specific subpopulation of hormonally null cells that are equally sensitive to the antiproliferative effects of pasireotide and octreotide, implicating SSTR2 receptors in this antimitotic response.

Effects of the Extract of a Chinese HerbTripterygium wilfordiiHook f on Rat Pituitary Gland

In China, the ethylacetate extract of the herb Tripterygium wilfordii Hook f (TWEE), containing the major active ingredient triptolide, is often used with favorable effect on rheumatoid arthritis patients, in alternation with the use of prednisone. The mechanism of this therapeutic effect, however, has not been completely delineated. In this study, we studied how TWEE and prednisone affect the pituitary and adrenal glands in rats. Thirty normal male Sprague-Dawley rats (ten per group) were randomly assigned to receive: (1) TWEE (25 mg/kg, twice a day), (2) prednisone (2 mg/kg, twice a day), or (3) vehicle (control) (0.5% sodium carboxymethyl cellulose 1 ml, twice a day), orally for 30 days. Pituitary and trunk blood were collected on day 31. Adrenocorticotropic hormone (ACTH) expression in the pituitary gland was assessed morphologically by immunohistochemical techniques. Plasma ACTH concentrations and serum corticosterone concentrations were quantitatively measured by radioimmunoassay. We found that TWEE significantly increased plasma ACTH concentration and serum corticosterone concentration and dramatically increased the number of ACTH-positive cells in the pituitary. Our findings indicate that TWEE can promote the synthesis and secretion of ACTH cells--in the pars distalis of the rat pituitary gland and the production of corticosterone in the zone fasciculata of the adrenal cortex. Our results indicate that TWEE has a cortical hormone-like function and can promote adrenal cortex function by activating the hypothalamus-pituitary-adrenal axis.

Galectin-3 Expression in Functioning and Silent ACTH-Producing Adenomas

Galectin-3 (Gal-3), a beta 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 folliculo-stellate (FS) cells and tumors and that Gal-3 had an important regulatory role in pituitary cell proliferation. 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.

Expression of 11 beta-hydroxysteroid dehydrogenase isoenzymes in the human pituitary: induction of the type 2 enzyme in corticotropinomas and other pituitary tumors.

One of the defining biochemical features of Cushing's disease is a relative insensitivity to glucocorticoid (GC) feedback, but an analysis of the GC receptor has failed to detect any major abnormalities. However, two isoenzymes of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD), either by converting cortisone (E) to cortisol (F) (type 1) or conversely by converting F to E (type 2), play an important prereceptor role in regulating corticosteroid hormone action at several sites. 11 beta HSD1 and -2 expression within the anterior pituitary gland itself may modulate GC feedback at an autocrine level, and we have speculated that this may be deranged in Cushing's disease. Detection of 11 beta HSD type 1 and 2 immunoreactive protein was performed using fluorescence immunohistochemistry. Double immunofluorescent studies were undertaken on normal pituitary to define the cellular localization of 11 beta HSD isoenzymes using antisera against GH, ACTH, LH, FSH, PRL, and S100, a nonhormonal marker of folliculo-stellate cells. In normal pituitary, positive staining for 11 beta HSD1-immunoreactive protein was observed in GH- and PRL-secreting cells and in folliculo-stellate cells; gonadotrophs, thyrotrophs, and ACTH-positive cells were negative. 11 beta HSD2 immunoreactivity was absent in all cell types. RT-PCR detected 11 beta HSD1 messenger ribonucleic acid (mRNA) expression in the normal pituitary; 11 beta HSD2 mRNA expression was also seen in most normal tissue. By contrast, in ACTH-secreting adenomas 11 beta HSD2 immunostaining was strongly positive in every case of corticotroph adenoma. 11 beta HSD1 immunoreactivity was also observed occasionally, but to a much lesser extent. In other pituitary tumors, both functional and nonfunctional, 11 beta HSD expression was variable in terms of isoenzyme mRNA and intensity of protein staining. The expression of 11 beta HSD1 (which generates F from E) in somatotrophs and lactotrophs suggests an autocrine role for this isoenzyme in the glucocorticoid regulation of pituitary GH and PRL secretion. 11 beta HSD2 expression is markedly induced in ACTH-secreting pituitary tumors and, by converting F to E, may explain the resetting of glucocorticoid feedback control in Cushing's disease.

Immunohistochemical survey of migration of human anterior pituitary cells in developmental, pathological, and clinical aspects: a review

Developmentally pathological conditions of the anterior pituitary cells include failed separation of the primary pituitary gland into sellar and pharyngeal ones, ectopic migration into the subarachnoid space, and basophil invasion into the posterior lobe although the last is a physiological phenomenon with pathological potentiality in certain circumstances. Pituitary primordium appears at about 4 weeks of gestation. One of the causes of the pituitary gland agenesis may be a formation of the primary hypothalamic ganglionic hamartoma just at the time of occurrence of the pituitary primordium, as analyzed in cases of Pallister-Hall syndrome. A double pituitary in a single individual is a rare malformation. Its pathogenesis is considered as a result of notochordal anomaly. In the 8th gestational week, the primary pituitary gland separates into sellar and pharyngeal parts. The disturbance of this histogenesis results in a rare pituitary malformation, a “pharyngosellar pituitary.” Despite the failed separation in this case, differentiation of the pituitary cells proceeds and the hormone production of this malformed pituitary gland can be displayed immunohistochemically. In this case, the distribution of the different hormone producing cells was atypical, particularly in those of gonadotropic hormones and ACTH. Life-long existence of the pharyngeal pituitary is a normal anatomical state in humans. Cell differentiation (hormone production) in the pharyngeal pituitary occurs about 4–10 weeks later than in the sellar pituitary. In pharyngeal pituitary, all kinds of adenohypophyseal hormones are produced. Extracranial pituitary adenomas (with intact sellar pituitary), exclusively found in the nasopharynx, sphenoid sinus, and clivus, may occur from the pharyngeal pituitary while another tumorigenesis can develop from the residual tissue fragment in the craniopharyngeal canal. The “overshoot” of the adenohypophyseal cell migration in the distal part of the sellar pituitary is frequently observed in the leptomeninges of the peri-infundibular or peri-hypothalamic region as ectopic pituitary cell clusters that are apparently independent of the pars tuberalis. It is suggested that these cells, frequently found in “normal” individuals, may be one of the possible origins of the intracranial ectopic pituitary adenomas. However, the reason why a majority of the reported intracranial ectopic pituitary tumors are ACTH-adenomas remains unexplained, since the ectopic cells, found in “normal” individuals, consist of fairly different hormone-producing cells. A further enigmatic phenomenon is a “basophil invasion.” ACTH-positive cells invade from the pars intermedia into the posterior lobe of the pituitary. This invasion increases in intensity and frequency according to increase in age. However, the invasion of ACTH cells is observed as early as in the fetal life. The invasive cells display occasionally cell atypia as well as mitotic activity. The origin of extremely rare pituitary adenomas inside the posterior lobe can be explained by the existence and proliferative activity of basophil invasion. Microsc. Res. Tech. 46:59–68, 1999. © 1999 Wiley-Liss, Inc.

Immunohistochemical survey of migration of human anterior pituitary cells in developmental, pathological, and clinical aspects: a review.

Developmentally pathological conditions of the anterior pituitary cells include failed separation of the primary pituitary gland into sellar and pharyngeal ones, ectopic migration into the subarachnoid space, and basophil invasion into the posterior lobe although the last is a physiological phenomenon with pathological potentiality in certain circumstances. Pituitary primordium appears at about 4 weeks of gestation. One of the causes of the pituitary gland agenesis may be a formation of the primary hypothalamic ganglionic hamartoma just at the time of occurrence of the pituitary primordium, as analyzed in cases of Pallister-Hall syndrome. A double pituitary in a single individual is a rare malformation. Its pathogenesis is considered as a result of notochordal anomaly. In the 8th gestational week, the primary pituitary gland separates into sellar and pharyngeal parts. The disturbance of this histogenesis results in a rare pituitary malformation, a "pharyngosellar pituitary." Despite the failed separation in this case, differentiation of the pituitary cells proceeds and the hormone production of this malformed pituitary gland can be displayed immunohistochemically. In this case, the distribution of the different hormone producing cells was atypical, particularly in those of gonadotropic hormones and ACTH. Life-long existence of the pharyngeal pituitary is a normal anatomical state in humans. Cell differentiation (hormone production) in the pharyngeal pituitary occurs about 4-10 weeks later than in the sellar pituitary. In pharyngeal pituitary, all kinds of adenohypophyseal hormones are produced. Extracranial pituitary adenomas (with intact sellar pituitary), exclusively found in the nasopharynx, sphenoid sinus, and clivus, may occur from the pharyngeal pituitary while another tumorigenesis can develop from the residual tissue fragment in the craniopharyngeal canal. The "overshoot" of the adenohypophyseal cell migration in the distal part of the sellar pituitary is frequently observed in the leptomeninges of the peri-infundibular or peri-hypothalamic region as ectopic pituitary cell clusters that are apparently independent of the pars tuberalis. It is suggested that these cells, frequently found in "normal" individuals, may be one of the possible origins of the intracranial ectopic pituitary adenomas. However, the reason why a majority of the reported intracranial ectopic pituitary tumors are ACTH-adenomas remains unexplained, since the ectopic cells, found in "normal" individuals, consist of fairly different hormone-producing cells. A further enigmatic phenomenon is a "basophil invasion." ACTH-positive cells invade from the pars intermedia into the posterior lobe of the pituitary. This invasion increases in intensity and frequency according to increase in age. However, the invasion of ACTH cells is observed as early as in the fetal life. The invasive cells display occasionally cell atypia as well as mitotic activity. The origin of extremely rare pituitary adenomas inside the posterior lobe can be explained by the existence and proliferative activity of basophil invasion.

Immunocytochemistry of ambiguous cells in adult and embryonic dwarf (dw) mouse pituitaries.

In the pars distalis of the pituitary gland in adult and embryonic dwarf (dw/dw) mutant mice, ambiguous cells exhibiting ultrastructural features common to growth hormone (GH) cells and prolactin (Prl) cells were analyzed by means of colloidal gold ultrastructural immunocytochemistry in order to define the functional nature of these peculiar cells. Adult and 18-day embryonic pituitaries from normal (+/+; dw/+) and dwarf (dw/dw) mice were processed with antibodies to GH, Prl, TSH (thyroid-stimulating hormone), ACTH (adrenocorticotropic hormone), LH (luteinizing hormone), FSH (follicle-stimulating hormone), and HCG (chorionic gonadotropic hormone). In the adult and embryonic dwarf pituitaries, the ambiguous cells reacted negatively to all of the antibodies except for anti-ACTH, which labeled them well. In addition, the ACTH-positive cells showed a much wider variety of shapes and granule size and distribution, as compared with normal adults. In the embryos, this variability in ACTH cell morphology occurred not only in dwarf embryos, but in their normal counterparts as well. The results thus suggest that adult dwarf pituitaries may retain an embryonic or incompletely differentiated form of ACTH cells.

Immunocytochemical observation of adenohypophysis in a human growth hormone (hGH) gene transgenic mouse

Adenohypophysis was immunocytochemically examined in an infertile female transgenic (Tg) mouse which carried human growth hormone (hGH) gene and had a high circulating level of hGH. No GH positive cells were detected. This confirmed the extrahypophyseal (ectopic) production of hGH and was coincident with the disappearance of parenchymal cells showing affinity to azocarmine in Azan staining. The normal frequency of ACTH positive cells was in accordance with the previous suggestion based on the changes found in zona fasciculata cells of the adrenal cortex. Most interesting findings were the detection of many PRL positive cells and the ovarian histology with nearly normal characteristics except for the presence of thick capsule and interstitial gland-like structure composed of large and light cells. Ovarian histology was also clearly different among individual Tg mice, even though they stemmed from the same line or progenitors, and had a similar phenotype. The current immunocytochemical observation well documented the changes revealed with Azan staining in the adenohypophysis about GH but not about PRL or ACTH. Thus, the immunocytochemical analysis of the adenohypophysis will provide useful methodology in assessment of endocrinological circumstances of Tg mice.