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Association of annexin V with prolactin in the rat anterior pituitary gland.

When pituitary extracts were subjected to non denaturing polyacrylamide gel electrophoresis, an unknown protein was found to associate with a proportion of the prolactin. This protein was dissociated from prolactin by sodium dodecyl sulfate. The protein was purified and sequenced. As the amino terminus was blocked, the amino acid sequences of three peptide fragments were determined. The obtained sequences of 41 amino acids were identical to partial sequences of a known protein, rat Annexin V. The molecular mass, 36 kDa, was also the same as the molecular weight of Annexin V. The existence of Annexin V mRNA in rat pituitary glands was also confirmed by polymerase chain reaction. These results show that Annexin V, a member of the calcium-dependent phospholipid binding proteins, is synthesized in the rat pituitary gland, and suggest its association with prolatin in the gland.

Animals↗

Genes that fashion the pituitary gland.

The pituitary gland with its distinct populations of hormone-producing cells is formed from precursor cells that are created when epithelial cells from the prospective diencephalon about the roof of the oral cavity during early development of the mammalian embryo. Successive steps of assembly during development lead from a rudimentary pouch to a definitive structure called Rathke's pouch, and finally to the pituitary gland with its anterior, intermediate and posterior lobes. The proliferation and differentiation of highly specialized cells from their precursors is directed by signaling cascades and transcriptional events that are being dissected by genetic and molecular approaches. Our current state of knowledge in this regard allows us to use the pituitary as a paradigm to delineate general principles of organogenesis in the mammalian organism.

Animals↗

Functional morphology of spontaneous hyperplastic and neoplastic lesions in the canine pituitary gland.

The pituitary glands of 10 male and 29 female dogs of different breeds had diffuse hyperplasia and hypertrophy of growth hormone or prolactin cells. Immunoreactive adrenocorticotrophin/melanotrophin was in the frequent focal hyperplastic lesions and micro- and macroadenomas of both pars distalis and pars intermedia of old female dogs. Pituitary tumours of other functional cell types were not found. Focal hyperplastic and neoplastic lesions usually were accompanied by spontaneous nodular hyperplasia or adenomas of the adrenal cortex as well as by changes in serum adrenocorticotrophic hormone and cortisol levels.

Adenoma↗

Melatonin receptors in the brain and pituitary gland of hypothalamo-pituitary disconnected Soay rams.

In Soay rams in which the pituitary gland has been surgically separated from the hypothalamus, blood prolactin concentrations vary in response to changes in photoperiod and the administration of melatonin, as in intact animals, providing evidence that melatonin acts within the pituitary gland to control prolactin secretion. In this study the presence of potentially functional melatonin receptors in the pars tuberalis and zona tuberalis (PT/ZT) of hypothalamo-pituitary disconnected (HPD) Soay rams is confirmed using both in vitro autoradiography with the ligand 2-(125I)-iodomelatonin and in situ hybridization for the melatonin receptor. There was no effect of the HPD operation on the pattern and quantity of 2-(125I)iodomelatonin binding in the brain demonstrating that this binding is independent of hypothalamic regulation. The possibility that melatonin may control prolactin secretion directly via specific receptors on lactotrophs was investigated using dual in situ hybridization with a (35S) labelled probe for the ovine melatonin receptor (Mel 1a(b)) and a Digoxigenin labelled probe for ovine prolactin. Melatonin receptor gene expression was observed in the PT/ZT in both intact and HPD rams, however, there was no colocalization with prolactin gene expression; only in the ZT was there a close association between cells expressing the melatonin receptor and lactotrophs. The results provide strong support for the view that melatonin acts via the PT/ZT to mediate the effects of photoperiod on the seasonal cycle in prolactin secretion.

Animals↗

Studies on the presence of vasopressin, oxytocin and vasotocin in the pineal gland, subcommissural organ and fetal pituitary gland: failure to demonstrate vasotocin in mammals.

The demonstration of vasotocin in the mammalian pineal gland, subcommissural organ and fetal pituitary gland by bioassay has led to hypotheses regarding the function of this hormone in various reproductive processes. Preliminary examinations of the pineal gland and subcommissural organ with a specific radioimmunoassay failed to show vasotocin immunoreactivity. The presence of vasotocin, vasopressin and oxytocin in the pineal gland, subcommissural organ and fetal neurohypophysis was therefore investigated, using three specific radioimmunoassays. Frog and chicken pituitary glands were used to validate the vasotocin radioimmunoassay. Direct measurements in diluted homogenates of pituitary glands from frogs, chickens, mid-term fetal sheep and near-term fetal seals revealed the presence of vasotocin only in the frog and chicken pituitary glands, while vasopressin and oxytocin were found in the two fetal pituitary homogenates. Vasopressin and ocytocin were measured in homogenates of rat and bovine pineal glands and in preparations of the subcommissural organ of rats and rabbits after extraction with Vycor glass powder, but no specific vasotocin immunoreactivity was observed. These results indicate a discrepancy between the reported biological activity of vasotocin in the pineal gland, subcommissural organ and fetal pituitary gland and the immunoreactivity of this material, which can at present only be explained by the presence of a peptide which is structurally closely related to, but not identical with, vasotocin.

Animals↗

Intercellular communication within the rat anterior pituitary gland: X. Immunohistocytochemistry of S-100 and connexin 43 of folliculo-stellate cells in the rat anterior pituitary gland.

Since Rinehart and Farquhar reported the presence of agranulated cells in the anterior pituitary gland in 1953, the functions of the folliculo-stellate cell remain to be clarified. Intercellular junctions have been described in the monkey, rat, and teleost anterior pituitary glands, indicating the existence of cell-to-cell communication within the organ. We pointed to their possible role in the rapid dissemination of information through a complex interconnecting system of follicles involving gap junctions. The gap junctional/folliculo-stellate cellular network was essential in the maturation and regulation of the pituitary gland system such as the hypothalamic-pituitary-gonadal axis. It has been was shown that a network participated in the conduction of electrophysiological information over a long distance using the ion Ca(++), which propagates to other folliculo-stellate cells by signaling through gap junctions. Sixty-day-old male rats were used in this study for light microscopic immunohistochemistry of S-100 protein, type I collagen, and connexin 43, and for electron microscopy to observe the morphological relationships between the cellular networks of folliculo-stellate cells and granulated pituitary cells. Clusters of anti-S-100 protein-positive cells were clearly observed in a region of the hypophysis tentatively named the transition zone. Anti-S-100 protein-positive cells and their cytoplasmic processes were also present in the anterior lobe and assembled together to form follicular lumina. Type I collagen was clearly shown outlining the incomplete lobular or ductule-like structure making cell cords in the anterior pituitary gland. Numerous microvilli were present within the follicular lumen while around the lumina, junctional specializations including gap junctions were positive for the connexin 43 protein. A nonuniform distribution of the connexin 43-positive sites were observed. Small or dot-shaped positive sites were noted where two clusters of cells were connected; the cells were identified as S-100 cells. Double immunohistochemical staining of the connexin 43 and growth hormone (GH) or connexin 43 and luteinizing hormone (LH) was also performed, demonstrating no direct relationship between the connexin 43 and either the GH or LH cells. These findings indicate that there are two kinds of messages necessary for the hormone release in the pituitary gland. One is via the portal vein system, the other is through the gap junction-mediated networks of folliculo-stellate cells. The granulated cells directly associate with cell membrane of folliculo-stellate cells are able to discharge secretory granules through communication via gap junctions, while those granulated cells that are more distant from the folliculo-stellate cells are only able to discharge hormones via the pituitary hormone-releasing hormone from the portal vein system.

Animals↗

Imaging of the pituitary gland in dogs with pituitary-dependent hyperadrenocorticism.

Detailed imaging of the pituitary gland is a prerequisite for a successful outcome of transsphenoidal hypophysectomy in dogs because it allows for accurate preoperative localization and assessment of the size of the pituitary gland. Cisternography allows assessment of even small increases in the height of the pituitary gland, but the magnitude of suprasellar expansion of pituitary tumors cannot be assessed with this technique. Large pituitary tumors with suprasellar expansion can readily be detected with conventional contrast-enhanced computed tomography (CT), while pituitary microadenomas can be localized with dynamic contrast-enhanced CT. Dynamic examination of the entire pituitary may be possible with spiral dynamic CT. Magnetic resonance imaging (MRI) of the canine pituitary gland provides a clear differentiation between the pituitary gland and the surrounding structures, but it remains to be investigated whether in dogs MRI can replace dynamic CT for the detection of microadenomas and whether it allows accurate localization of the pituitary gland relative to the surgical landmarks.

Adrenocortical Hyperfunction↗

Inhibition by testosterone of prolactin and growth hormone release from chicken anterior pituitary glands in vitro.

Pituitary glands and hypothalami from broiler fowl were incubated in medium containing testosterone, and prolactin and GH release were determined. Pituitary glands were also preincubated for 20 h in medium containing testosterone, and then in medium containing various secretagogues. Testosterone inhibited the release of prolactin directly from the pituitary gland in a concentration-related manner. The hypothalamus stimulated the release of prolactin, but by a lesser amount in the presence of testosterone. When pituitary glands were preincubated with testosterone, subsequent release of prolactin was inhibited, except with the highest concentration which stimulated prolactin release. Hypothalamic extract (HE) markedly stimulated prolactin release from control pituitary glands although testosterone-primed glands were less responsive. The stimulation of prolactin release by thyrotrophin releasing hormone (TRH) and prostaglandin E2 (PGE2) was also reduced by preincubation of the pituitary glands with testosterone. Priming with testosterone did not affect the release of GH from pituitary glands alone, but reduced the TRH-, HE- and PGE2-stimulated release of GH. These results demonstrate that testosterone directly inhibits prolactin secretion and reduces the sensitivity of pituitary lactotrophs and somatotrophs to provocative stimuli.

Animals↗

MR-appearance of the pituitary gland before and after resection of pituitary macroadenomas.

Pituitary macroadenomas can cause severe compression and displacement of the pituitary gland. This study was undertaken to determine the value of contrast administration for the detection of the pituitary gland in patients with pituitary macroadenomas, to evaluate the preoperative location and configuration of the pituitary gland and to describe its postoperative changes after tumour resection. Preoperative and postoperative MR imaging examinations of 30 patients with histologically proven pituitary macroadenomas were retrospectively evaluated. The examinations were performed on a 1.5 T unit, by obtaining T1-weighted sagittal and coronal images after administration of gadopentetate dimeglumine. On preoperative MR images, contrast administration increased the detectability of the anterior lobe from 30% to 80%. Depending on the size and extension of the adenoma, the pituitary gland was displaced to the suprasellar space (53%) and/or deformed to a club-shaped (27%) or sickle-shaped (47%) configuration. In six patients, the sickle-shaped pituitary gland was interposed between the cavernous sinus and the adenoma ('rim-sign'), which was seen only on gadopentetate dimeglumine-enhanced images. In these cases, there was no infiltration of the cavernous sinus at surgery. Postoperatively, descent of the pituitary gland was found in 63%, and reexpansion in 54%. We conclude that contrast administration improves the detectability of the pituitary gland on preoperative MR images, and that the displacement and deformation of the pituitary gland depend on the size, location and extension of the adenoma. Preoperatively, demonstration of the pituitary gland interposed between the cavernous sinus and the adenoma ('rim-sign') is a very useful sign for exclusion of cavernous sinus infiltration, best seen on contrast-enhanced coronal MR images. The normal postoperative changes of the pituitary gland after tumour resection include repositioning and re-expansion.

Adenoma↗

Cellular distribution and gene regulation of estrogen receptors alpha and beta in the rat pituitary gland.

The pituitary gland is a heterogeneous tissue comprised of several hormone secreting and supporting cells, most of which are targeted by estrogens. Estrogen-induced changes in the pituitary are presumably mediated via the classical estrogen receptor, ER alpha. However, a novel receptor, ER beta, and pituitary-specific truncated estrogen receptor products (TERPs) were recently identified. The objectives of this study were to examine the distribution of these receptors in the rat pituitary and compare their regulation by estradiol in Sprague-Dawley and the estrogen-sensitive Fischer 344 rats. Pituitary cryosections were subjected to immunocytochemistry for specific cell types, followed by in situ hybridization for ER alpha or ER beta. ER alpha was expressed by approximately 45% of the lactotrophs and melanotrophs, 35% of the corticotrophs and folliculo-stellate cells, and 25% of the gonadotrophs. The expression of ER beta showed a similar pattern but was generally lower than ER alpha. In two cell types, melanotrophs and gonadotrophs, ER beta expression was significantly lower than ER alpha. In the second experiment, pituitary sections were immunostained for ER alpha, followed by in situ hybridization for ER beta. Only a minute population (6-10%) of either anterior or intermediate lobe cells coexpressed ER alpha and ER beta. In the next experiment, Fischer 344 and Sprague-Dawley rats were injected with oil or estradiol for 24 h. Total RNA from dissected anterior and posterior (neurointermediate) pituitaries was subjected to RT-PCR for ER alpha, ER beta, or TERPs. Interestingly, ER alpha and ER beta were unchanged by estradiol in either lobe of the pituitary. In contrast, estradiol increased pituitary TERP messenger RNA levels 4- to 7-fold. A 20-kDa TERP protein was detected by Western blots in the pituitary but not the uterus. There were no differences in the estradiol-induced expression of any of the receptors between the two strains of rats. We conclude that: 1) ER beta is expressed in all anterior and intermediate lobe cell types examined, albeit at a lower level than ER alpha; 2) no more than 10% of pituitary cells coexpress ER alpha and ER beta; and 3) estradiol markedly increases TERP messenger RNA levels but does not alter the expression of ER alpha or ER beta. We propose that estrogen receptor heterogeneity contributes to the diversity of pituitary cell responsiveness to estrogens.

Animals↗

Estrogen regulation of peptidylglycine alpha-amidating monooxygenase expression in anterior pituitary gland.

The pituitary is a rich source of peptidylglycine alpha-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase catalytic domains necessary for the two-step formation of alpha-amidated peptides from their COOH-terminal glycine extended precursors. Expression of PAM was evaluated in the anterior pituitary of intact cycling adult female rat and after experimental manipulation of estrogen status. PAM messenger RNA (mRNA) levels showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17 beta-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. In situ hybridization of anterior pituitary sections using 35S-labeled full length RNA antisense transcripts of rat PAM-1 complementary DNA showed that 17 beta-estradiol treatment induced an overall decrease of the hybridization signal, as compared with OVX rats. Progesterone treatment did not change PAM mRNA levels both in OVX or OVX + E2 rats. Based on Northern blot analysis and amplification of fragments derived from rat PAM-1 by RT-PCR, it was found that estrogen status does not affect the distribution of PAM mRNA among its various alternatively spliced forms. In OVX 17 beta-estradiol treated rats, the specific activity of PAM in the anterior pituitary decreased in both soluble and particulate fractions compared with OVX animals. Western blot analysis demonstrated a 105-kDa PAM protein in particulate fractions prepared from OVX and OVX-17 beta-estradiol treated animals. The soluble fraction from OVX animals contained major PAM proteins of 105, 95, 84, 75, and 45 kDa, and 17 beta-estradiol treatment caused a decrease in the prevalence of these proteins. These results indicate that estrogens are involved, either directly or indirectly, in regulating the expression of PAM in several cell types in the anterior pituitary gland.

Alternative Splicing↗

Identification of a novel keratinocyte mitogen derived from bovine pituitary glands.

Bovine pituitary glands contain one or more factors that are important for keratinocyte proliferation in serum-free culture medium. We used a tissue culture system in which the growth of keratinocytes in basal medium (KBM) containing insulin was dependent upon exogenous growth factors. Using this experimental system, we began to purify and characterize the pituitary factor(s) necessary for clonal growth of human keratinocytes in serum-free medium. Proteins of approximately 150 kDa and 95 kDa bound specifically to living keratinocytes, and we suggest that the 95 kDa protein is a likely novel mitogen. Although prolactin has been previously identified as a pituitary hormone that may act as an in vitro mitogen for keratinocytes, imunoblots indicated that the 95 kDa protein was unrelated to prolactin. Furthermore, the 95 kDa protein showed high homology with a bovine 90 kDa heat shock protein in the limited sequencing of an internal peptide.

Amino Acid Sequence↗