Prolactin-producing Cells Research Articles

Conditional Pten inactivation in pituitary results in sex-specific prolactinoma formation

Pituitary tumors, including prolactinomas, present significant clinical challenges that require a deeper understanding of their molecular roots for improved diagnostics and therapies. Here, we investigate the role of the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K) pathway in pituitary tumorigenesis using a mouse model. Conditional knockout of Pten in all pituitary cell lineages resulted in prolactinoma formation exclusively in female mice, demonstrating the critical role of PTEN in pituitary homeostasis. While Pten inactivation induced Akt activation in all pituitary cells, only prolactin-producing cells exhibited tumorigenic changes, suggesting specific cell-type effects. Histological and molecular analyses of prolactinomas revealed similarities with human pituitary tumors, such as decreased vascularization and cell adhesion proteins and increased accumulation of cell cycle proteins. Notably, prolactinomas displayed diminished levels of phosphorylated extracellular signal-regulated kinase (ERK), implicating downregulation of ERK in tumorigenesis. Finally, we analyzed PTEN/PI3K activation in a collection of human pituitary tumors. Overall, our study delineates the intricate interplay between the PTEN and ERK signaling pathways, providing insights into sex-specific mechanisms of pituitary tumorigenesis and potential therapeutic strategies for prolactinomas.

7883 Atypical Presentation of Macroprolactinoma: Discordantly Low Serum Prolactin and Therapeutic Challenges

Abstract Disclosure: S. Hossain: None. M.S. Hossain: None. B. Gautam: None. S.C. Kumar: None. H. Liao: None. D.S. Rosenthal: None. Introduction: Pituitary tumors are categorized as microadenomas (<1 cm) and macroadenomas (>1 cm) with prolactinomas being one of the most common causes. The mean prevalence of prolactinomas is 10 per 100,000 in men and 30 per 100,000 in women. Serum prolactin levels corresponds to the size of the prolactinoma and any discrepancy should be treated with caution as that may indicate co-existence of pluripotent cells. We present a patient who presented with visual field defect and headaches, found to have a pituitary macroadenoma with discordantly low serum prolactin level compared to the size of adenoma, prompting a change in the treatment course. Case Presentation: A 42 year old male with no past medical history was seen as an endocrinology consult requested by neurosurgery for frequent headaches and visual problems for 1 year. Vital signs were normal, with negative orthostatic drop of blood pressure. He had prominent bitemporal hemianopsia confirmed by formal visual field testing by Ophthalmology. No galactorrhea or features suggestive of acromegaly or hyperthyroidism. He complained of erectile dysfunction but appeared to have normal male secondary sexual characteristics. MRI of brain showed a suprasellar mass measuring 3.8 x 2.6 x 2.2 cm with mass effect on the optic chiasm, hypothalamus with extension and infiltration into the right cavernous sinus. Lab results showed elevated prolactin of 1343.5 ng/ml confirmed by serial dilution, low serum testosterone level of 134 ng/dl and inappropriately normal FSH and LH. Complete blood counts, comprehensive metabolic panel, TFT, ACTH, cortisol and IGF-1 were normal. Patient was started on bromocriptine and he noticed improvement in his field of vision and acuity within one week of starting therapy. He continued to show sustained improvement in vision and resolution of his headaches at the one-month mark. MRI 2 months from starting bromocriptine showed modest reduction in size of the tumor to 2.1 x 2.3 x 2.4 cm with continued mass effect on the optic chiasm. Prolactin level decreased to 69.3 ng/ml. Therefore, although there was some improvement in visual fields, surgical resection was recommended to alleviate the mass effect on the optic chiasm. Conclusion: Our case highlights the significance of identifying discrepancies between the size of a prolactinoma and serum prolactin levels. Expected prolactin level from a prolactinoma similar in size to our case would be >3000 ng/ml when ours was 1343.5ng/ml. Despite undergoing treatment and lowering of prolactin levels our patient's pituitary adenoma did not significantly reduce in size after two months of treatment with bromocriptine and the mass continued to exert pressure on the optic chiasm. This suggests possible presence of pluripotent cells in addition to prolactin-producing cells within the tumor. The recommended course of action was surgical resection of the mass. Presentation: 6/1/2024

Morphological and Functional Changes of Pituitary GH and PRL Cells Following Prolonged Exposure of Female Rats to Constant Light

Abstract Light regulates numerous physiological functions including secretion of different hormones. Our aim was to determine morphological and functional changes of the pituitary growth hormone (GH) and prolactin (PRL) producing cells in female rats exposed to constant light regime from the peripubertal to adult period of life. Starting from the thirtieth postnatal day, female Wistar rats were exposed to constant light (600 lx) for the following 95 days. Controls were maintained under the regular laboratory lighting conditions. The GH and PRL cells were immunohistochemically visualized. Changes in cell volumes and volume densities were evaluated by stereology. Concentrations of PRL and GH in circulation were also determined. We detected significant decrease of the GH cell volume and volume density, followed by reduced the GH blood concentration in comparison to the controls. In contrast, PRL cells were larger in size and their volume density was significantly increased when compared to the controls. Accordingly, PRL concentration was elevated. It can be concluded that exposure of female rats to constant light regime, from peripubertal to adult period of life, causes inhibition of the pituitary GH and stimulation of PRL cells.

Fluctuation of CD9/SOX2-positive cell populations during the turnover of GH- and TSH-producing cells in the adult anterior pituitary gland.

The adenohypophysis is comprised of the anterior and intermediate lobes (AL and IL, respectively). Cluster of differentiation 9 (CD9)- and sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor hormone-producing cells in the AL. They are located in the marginal cell layer (MCL) facing Rathke's cleft between the AL and IL (primary niche) and the parenchyma of the AL (secondary niche). We previously showed that, in rats, CD9/SOX2-positive cells in the IL side of the MCL (IL-side MCL) migrate to the AL side (AL-side MCL) and differentiate into prolactin-producing cells (PRL cells) in the AL parenchyma during pregnancy, lactation, and diethylstilbestrol treatment, all of which increase PRL cell turnover. This study examined the changes in CD9/SOX2-positive stem/progenitor cell niches and their proportions by manipulating the turnover of growth hormone (GH)- and thyroid-stimulating hormone (TSH)-producing cells (GH and TSH cells, respectively), which are Pit1 lineage cells, as well as PRL cells. After induction, the isolated CD9/SOX2-positive cells from the IL-side MCL formed spheres and differentiated into GH and TSH cells. We also observed an increased GH cell proportion upon treatment with GH-releasing hormone and recovery from continuous stress and an increased TSH cell proportion upon propylthiouracil treatment, concomitant with alterations in the proportion of CD9/SOX2-positive cells in the primary and secondary niches. These findings suggest that CD9/SOX2-positive cells have the potential to supply GH and TSH when an increase in GH and TSH cell populations is required in the adult pituitary gland.

Functional and developmental heterogeneity of pituitary lactotropes in medaka

In fish, prolactin-producing cells (lactotropes) are located in the anterior part of the pituitary and play an essential role in osmoregulation. However, small satellite lactotrope clusters have been described in other parts of the pituitary in several species. The functional and developmental backgrounds of these satellite clusters are not known. We recently discovered two distinct prolactin-expressing cell types in Japanese medaka (Oryzias latipes), a euryhaline species, using single cell transcriptomics. In the present study, we characterize these two transcriptomically distinct lactotrope cell types and explore the hypothesis that they represent spatially distinct cell clusters, as found in other species. Single cell RNA sequencing shows that one of the two lactotrope cell types exhibits an expression profile similar to that of stem cell-like folliculo-stellate cell populations. Using in situ hybridization, we show that the medaka pituitary often develops additional small satellite lactotrope cell clusters, like in other teleost species. These satellite clusters arise early during development and grow in cell number throughout life regardless of the animal’s sex. Surprisingly, our data do not show a correspondence between the stem cell-like lactotropes and these satellite lactotrope clusters. Instead, our data support a scenario in which the stem cell-like lactotropes are an intrinsic stage in the development of every spatially distinct lactotrope cluster. In addition, lactotrope activity in both spatially distinct lactotrope clusters decreases when environmental salinity increases, supporting their role in osmoregulation. However, this decrease appears weaker in the satellite lactotrope cell clusters, suggesting that these lactotropes are regulated differently.

Anatomy and histology of the Göttingen minipig adenohypophysis with special emphasis on the polypeptide hormones: GH, PRL, and ACTH.

The pituitary is involved in the regulation of endocrine homeostasis. Therefore, animal models of pituitary disease based on a thorough knowledge of pituitary anatomy are of great importance. Accordingly, we aimed to perform a qualitative and quantitative description of polypeptide hormone secreting cellular components of the Göttingen minipig adenohypophysis using immunohistochemistry and stereology. Estimates of the total number of cells immune-stained for adrenocorticotrophic hormone (ACTH), prolactin (PRL), and growth hormone (GH) were obtained with the optical fractionator technique using Stereo Investigator software. Moreover, 3D reconstructions of cell distribution were made. We estimated that the normal minipig adenohypophysis contains, on average, 5.6 million GH, 3.5 million PRL, and 2.4 million ACTH producing cells. The ACTH producing cells were widely distributed, while the PRL and GH producing cells were located in clusters in the central and lateral regions of the adenohypophysis. The morphology of the hormone producing cells also differs. We visualized a clear difference in the numerical density of hormone producing cells throughout the adenohypophysis. The relative proportions of the cells analyzed in our experiment are comparable to those observed in humans, primates, and rodents; however, the distribution of cells differs among species. The distribution of GH cells in the minipig is similar to that in humans, while the PRL and ACTH cell distributions differ. The volume of the pituitary is slightly smaller than that of humans. These data provide a framework for future large animal experimentation on pituitary function in health and disease.

CD9-positive cells in the intermediate lobe of the pituitary gland are important supplier for prolactin-producing cells in the anterior lobe.

A supply of hormone-producing cells from stem/progenitor cells is critical to sustain the endocrine activity of the pituitary gland. In the adenohypophysis composing the anterior and intermediate lobe (AL and IL, respectively), stem/progenitor cells expressing sex-determining region Y-box 2 (SOX2) and S100β are located in the marginal cell layer (MCL) facing Rathke's cleft (primary niche) and the parenchyma of the AL (secondary niche). Our previous studies using mice and rats indicated that the tetraspanin superfamily CD9 and CD81 are expressed in S100β/SOX2-positive cells of primary and secondary niches (named CD9/CD81/S100β/SOX2-positive cell), and the cells located in the AL-side niches exhibit plasticity and multipotency. However, it is unclear whether CD9/CD81/S100β/SOX2-positive cells in the IL-side primary niche are stem/progenitor cells for the AL or IL. Here, we successfully isolated pure CD9/CD81/S100β/SOX2-positive cells from the IL-side primary niche. They had a higher level of S100β and SOX2 mRNA and a greater pituisphere forming capacity than those of CD9/CD81/S100β/SOX2-positive cells isolated from the AL. They also had capacity to differentiate into all types of adenohypophyseal hormone-producing cells, concomitantly with the loss of CD9 expression. Loss of CD9 and CD81 function in CD9/CD81/S100β/SOX2-positive cells by siRNA treatment impaired prolactin cell differentiation. Consistently, in the pituitary gland of CD9/CD81 double knockout mice, dysgenesis of the MCL and a lower population of prolactin cells were observed. These results suggest that the CD9/CD81/S100β/SOX2-positive cells in the MCL of the IL-side are potential suppliers of adult core stem cells in the AL.

Gestational dioxin exposure suppresses prolactin-stimulated nursing in lactating dam rats to impair development of postnatal offspring

A number of epidemiological studies have implicated environmental chemicals including dioxins in the induction of negative effects on child development. To clarify the underlying mechanisms, almost all toxicologists have concentrated on effects on the offspring themselves. We examined an alternative hypothesis that gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a highly-toxic dioxin, targets factors related to maternal childcare to disturb offspring development. Oral administration of TCDD (1 µg/kg) to pregnant rats on gestational day 15 suppressed maternal licking behavior, a nursing behavior, and mammary gland maturation during the lactational stage, as well as the body weight and short-term memory of postnatal offspring. In support of these findings, maternal production of prolactin, a pituitary hormone essential for nursing including milk production, was decreased during the same period. Intracerebroventricular infusion of prolactin to dioxin-exposed dams restored or tended to restore many of the above defects observed both in mothers and offspring. The TCDD-dependent defects in maternal nursing behaviors can be due to a direct action on aryl hydrocarbon receptor (AHR) of lactating dams, because they did not emerge in AHR-knockout dams or control dams with TCDD-exposed offspring. Further examinations revealed that TCDD induces transforming growth factor β1 expression, which suppresses prolactin-producing cell proliferation, in a nursing period-specific manner. In agreement with this, the number of prolactin-positive cells in nursing dams was decreased by TCDD. These results provide novel evidence that gestational dioxin exposure attenuates prolactin-stimulated nursing in lactating dams to impair offspring development, and that immaturity of prolactin-producing cells can contribute to them.

Seawater transfer down-regulates C-type natriuretic peptide-3 expression in prolactin-producing cells of Japanese eel: Negative correlation with plasma chloride concentration

In euryhaline fishes, atrial and B-type natriuretic peptides are important hormones in hypo-osmoregulation, whereas osmoregulatory functions of C-type natriuretic peptides (CNPs) remain to be investigated. Although four CNP isoforms (CNP1-4) are mainly expressed in the brain, multiorgan expression of CNP3 was found in euryhaline Japanese eel, Anguilla japonica. Here we identified the CNP3-expressing cells and examined their response to osmotic stress in eel. CNP3 was expressed in several endocrine cells: prolactin-producing cells (pituitary), glucagon-producing cells (pancreas), and cardiomyocytes (heart). Pituitary CNP3 expression was the highest among organs and was decreased following seawater transfer, followed by a decrease in the freshwater-adaptating (hyper-osmoregulatory) hormone prolactin. We also showed the negative correlation between CNP3/prolactin expression in the pituitary and plasma Cl− concentration, but not for plasma Na+ concentration. These results suggest that CNP3 in the pituitary (and pancreas) plays a critical role in freshwater adaptation of euryhaline eel together with prolactin.

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Is Highly Expressed in Mouse Tissues With Metabolic Function.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) form a family of atypical growth factors discovered for their neuroprotective properties in the central nervous system (CNS) in animal models of neurodegenerative diseases. Although their mechanism of protective action still remains unclear, it has been suggested that both MANF and CDNF promote cell survival through regulating the unfolded protein response (UPR), thereby relieving endoplasmic reticulum (ER) stress. Recent studies identified MANF for its emerging roles in metabolic function, inflammation and pancreatic β-cells. We have found that MANF deletion from the pancreas and β-cells leads to postnatal depletion of β-cells and diabetes. Moreover, global MANF-deficiency in mice results in severe diabetes-independent growth retardation. As the expression pattern of MANF in mouse tissues has not been extensively studied, we set out to thoroughly investigate MANF expression in embryonic and adult mice using immunohistochemistry, histochemical X-gal staining, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription PCR (RT-qPCR). We found that MANF is highly expressed in brain neurons regulating energy homeostasis and appetite, as well as in hypothalamic nuclei producing hormones and neuropeptides important for different body functions. Strong expression of MANF was also observed in peripheral mouse tissues and cells with high secretory and metabolic function. These include pituitary gland and interestingly we found that the anterior pituitary gland is smaller in MANF-deficient mice compared to wild-type mice. Consequently, we found reduction in the number of growth hormone- and prolactin-producing cells. This combined with increased expression of UPR genes, reduced number of proliferating cells in the anterior pituitary and dysregulated expression of pituitary hormones might contribute to the severe growth defect seen in the MANF knockout mice. Moreover, in this study we compared MANF and CDNF levels in mouse tissues. Unlike MANF, CDNF protein levels are generally lower in mouse tissues, and the highest levels of CDNF was observed in the tissues with high-energy demands and oxidative roles, including heart, muscle, testis, and brown adipose tissue.

Frequent Detection of Pituitary-Derived PrPres in Human Prion Diseases.

Human prion diseases including sporadic Creutzfeldt-Jakob disease (sCJD), inherited prion diseases, and acquired human prion diseases are lethal neurodegenerative diseases. One of the major sources of iatrogenic Creutzfeldt-Jakob disease was human growth hormone (hGH-iCJD) derived from contaminated cadaveric pituitaries. The incidence of hGH-iCJD has decreased since changing from growth hormone extracted from human cadaveric pituitaries to recombinant pituitary hormones. However, extensive analysis on the localization and detecting of abnormal prion protein in the pituitary gland are limited. In this study, we examined 9 autopsied brains and pituitary glands from 6 patients with prion disease (3 Gerstmann-Sträussler-Scheinker disease, 2 sCJD, and 1 dura mater graft-associated CJD) and 3 individuals with nonprion diseases. Western blot analysis of pituitary samples demonstrated unique glycoforms of normal cellular prion protein with molecular weights of 30-40 kDa, which was higher than the typical 25-35 kDa prion protein in brains. Proteomic analysis also revealed prion protein approximately the molecular weight of 40 kDa in pituitary samples. Moreover, proteinase K-resistant Prion protein was frequently detected in pituitary samples of the prion diseases. Immunohistochemistry for Prion protein revealed mosaic cellular distribution preferentially in growth hormone- or prolactin-producing cells.

Role of kisspeptin and Kiss1R in the regulation of prolactin gene expression in rat somatolactotroph GH3 cells.

Hypothalamic kisspeptin is a known principal activator of gonadotropin-releasing hormone neurons and governs the hypothalamic-pituitary-gonadal axis. Previous reports have shown that kisspeptin is also released into the hypophyseal portal circulation and directly affects the anterior pituitary. In this study, we examined the direct effect of kisspeptin on pituitary prolactin-producing cells. The rat pituitary somatolactotroph cell line GH3 expresses the kisspeptin receptor (Kiss1R); however, in these cells, kisspeptin failed to stimulate prolactin-promoter activity. When GH3 cells overexpressed Kiss1R, kisspeptin clearly increased prolactin-promoter activity, with a concomitant increase in extracellular signal-regulated kinase (ERK) and cAMP/protein kinase A (PKA) signaling pathways. In the experiments using GH3 cells overexpressing Kiss1R, kisspeptin did not potentiate thyrotropin-releasing hormone (TRH)-induced prolactin-promoter activity, but it potentiated the pituitary adenylate cyclase-activating polypeptide-induced prolactin-promoter activity, with a concomitant enhancement of ERK and PKA signaling pathways. Although the basal and TRH-induced prolactin-promoter activities were not modulated by increasing amounts of Kiss1R expression in GH3 cells, kisspeptin-stimulated prolactin-promoter activity was increased by the amount of Kiss1R overexpression. Endogenous Kiss1r mRNA expression in GH3 cells was significantly increased by treatment with estradiol (E2) but not by TRH. In addition, kisspeptin's ability to stimulate prolactin-promoter activity was restored after E2 treatment in non-transfected GH3 cells. Our current observations suggest that kisspeptin might have a direct effect on prolactin expression in the anterior pituitary prolactin-producing cells under the influence of E2, which may regulate Kiss1R expression and function.

ALK7 expression in prolactinoma is associated with reduced prolactin and increased proliferation.

Prolactinoma represents the most frequent hormone-secreting pituitary tumours. These tumours appear in a benign form, but some of them can reach an invasive and aggressive stage through an unknown mechanism. Discovering markers to identify prolactinoma proliferative and invading character is therefore crucial to develop new diagnostic/prognostic strategies. Interestingly, members of the TGFβ-Activin/BMP signalling pathways have emerged as important actors of pituitary development and adult function, but their role in prolactinomas remains to be precisely determined. Here, using a heterotopic allograft model derived from a rat prolactinoma, we report that the Activins orphan type I receptor ALK7 is ectopically expressed in prolactinomas-cells. Through immunohistological approaches, we further confirm that normal prolactin-producing cells lack ALK7-expression. Using a series of human tumour samples, we show that ALK7 expression in prolactinomas cells is evolutionary conserved between rat and human. More interestingly, our results highlight that tumours showing a robust expression of ALK7 present an increased proliferation as address by Ki67 expression and retrospective analysis of clinical data from 38 patients, presenting ALK7 as an appealing marker of prolactinoma aggressiveness. Beside this observation, our work pinpoints that the expression of prolactin is highly heterogeneous in prolactinoma cells. We further confirm the contribution of ALK7 in these observations and the existence of highly immunoreactive prolactin cells lacking ALK7 expression. Taken together, our observations suggest that Activin signalling mediated through ALK7 could therefore contribute to the hormonal heterogeneity and increased proliferation of prolactinomas.

Mechanisms regulating angiogenesis underlie seasonal control of pituitary function

Seasonal changes in mammalian physiology, such as those affecting reproduction, hibernation, and metabolism, are controlled by pituitary hormones released in response to annual environmental changes. In temperate zones, the primary environmental cue driving seasonal reproductive cycles is the change in day length (i.e., photoperiod), encoded by the pattern of melatonin secretion from the pineal gland. However, although reproduction relies on hypothalamic gonadotrophin-releasing hormone output, and most cells producing reproductive hormones are in the pars distalis (PD) of the pituitary, melatonin receptors are localized in the pars tuberalis (PT), a physically and functionally separate part of the gland. How melatonin in the PT controls the PD is not understood. Here we show that melatonin time-dependently acts on its receptors in the PT to alter splicing of vascular endothelial growth factor (VEGF). Outside the breeding season (BS), angiogenic VEGF-A stimulates vessel growth in the infundibulum, aiding vascular communication among the PT, PD, and brain. This also acts on VEGF receptor 2 (VEGFR2) expressed in PD prolactin-producing cells known to impair gonadotrophin secretion. In contrast, in the BS, melatonin releases antiangiogenic VEGF-Axxxb from the PT, inhibiting infundibular angiogenesis and diminishing lactotroph (LT) VEGFR2 expression, lifting reproductive axis repression in response to shorter day lengths. The time-dependent, melatonin-induced differential expression of VEGF-A isoforms culminates in alterations in gonadotroph function opposite to those of LTs, with up-regulation and down-regulation of gonadotrophin gene expression during the breeding and nonbreeding seasons, respectively. These results provide a mechanism by which melatonin can control pituitary function in a seasonal manner.

Abstract 3487: Alcohol programs the pituitary to produce aggressive prolactin-producing tumors

Abstract Chronic alcohol consumption is known to increase prolactin levels in blood and cell proliferation in pituitary prolactin-producing cells known as lactotropes, resulting in hyperprolactinemia in human and animals. We have recently shown that alcohol abuse during pregnancy result in fetal programming that alters lactotropic cell production of prolactin in the offspring during the adulthood. We determined whether fetal alcohol exposure increases the susceptibility to estrogen-induced pituitary prolactin-secreting tumors (prolactinomas). Pregnant Fischer 344 rats were fed between gestational days 7 and 21 with a liquid diet containing alcohol (AF), pair-fed with isocaloric liquid diet (PF), or fed ad libitum with rat chow (AD). At 90 days of age, female offspring rats were ovariectomized and received a subcutaneous estradiol implant. These rats were sacrificed at 3 moths after the estradiol implants. At the time of sacrifice, pituitaries of these animals were inspected for tumor and whole body were inspected for any tumor metastasis. Estradiol treatment increased pituitary weight about 5-folds in AD and PF treated groups and increased about 30-folds in the AF treated group. Most tumors in the AF group were hemorrhagic. About 30% AF rats had some tumors in the non-pituitary sites. AD and PF rats did not show any non-pituitary site tumor. Histopathological evaluation revealed that tumors in AF group were more densely packed cells as compare to the PF and AD groups who showed uniform cells with abundant cytoplasm. Pituitary tumor from AF animals showed strong nuclear p53, Ki67 and prolactin immunoreactivity. When AF rat pituitaries were grown in cultures, cells rapidly grew and formed colonies. Colony formation and rapid growth rate were not observed in cells of the pituitary from PF and AD rats. When pituitary tumor cells of AF-treated pituitary, but not AD- or PF-treated pituitaries were grown in ultra-low attachment plate, spheres developed. These spheres expressed genes and proteins related to multipotency (OCT4, NANOG, KLF4, SOX2, Nestin, CD34 and CD133). Differentiated cells from pituitary tumorspheres of AF animals successfully induce tumor in scid mice after three weeks from cells transplantation. In addition, pituitary tumor from AF animals showed strong immunoreactivity for N-cadherin and Snail, two epithelial-mesenchymal transitions (EMT) factors. Furthermore, RNA-seq of genes in tumor pituitaries of AF and AD rats revealed 8782 genes are significantly changed in AF compared to AD P<0.01. Many of these genes were involved in the cell migration, actin cytoskeletal regulation and cell-cell junction formations which are the most important phenotypes occur during EMT. These data provide evidence for the possible development of aggressive prolactinoma development in the pituitary after estrogen treatment in fetal alcohol exposed female rats. (This work is supported by a National Institute of Health grant R01 AA11591). Citation Format: Shaima Jabbar, Jinhee Park, William Belden, Dipak K. Sarkar. Alcohol programs the pituitary to produce aggressive prolactin-producing tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3487.

Effects of Estradiol-17β or Dihydrotestosterone on Cell Types in Equine Pituitaries Staining for Prolactin, Growth Hormone, or Both

Ovariectomized pony mares were treated with vehicle (controls), estradiol-17β (estradiol), or dihydrotestosterone (DHT) daily for 21 days in summer, and the pituitary content of prolactin and growth hormone (GH) and the numbers of cells staining immunocytochemically for prolactin (lactotropes), GH (somatotropes), or both hormones (mammosomatotropes) were assessed. Treatments did not alter (P > .1) plasma prolactin concentrations over the 21 days. Estradiol treatment increased (P < .05) the pituitary content of prolactin several-fold relative to controls and DHT-treated ponies. The number of lactotropes was also increased (P < .05), as was the number of mammosomatotropes (P < .05). Treatment with DHT did not alter (P > .1) any prolactin characteristic. The number of somatotropes was not altered by either treatment (P > .1) nor was the pituitary content of GH (P > .1). Given that the mass of prolactin accumulation in the pituitary in response to estradiol was several-fold greater than the approximate doubling of the number of lactotropes and mammosomatotropes, it appears that estradiol administration to ovariectomized pony mares stimulates not only the number of prolactin-producing cells but also the amount of prolactin production per cell.

Lactotrophs: the new and major source for VEGF secretion and the influence of ECM on rat pituitary function in vitro.

Vascular endothelial growth factor (VEGF) plays a pivotal role in pituitary endocrine function by influencing fenestration and blood vessel growth. Folliculostellate (FS) cells, which represent only a small number of pituitary cells, are recognized to produce VEGF. Tissue sections and primary pituitary cell cultures from rat pituitary glands were performed to co-localize VEGF and pituitary lactotrophs, which represents nearly 50% of all pituitary cells, by immunofluorescence. VEGF is co-localized with prolactin-producing cells in vivo and in vitro. FS cells are present infrequently in vivo (1.6%) and in vitro (2.4%). Culture supernatants were analyzed for the presence of VEGF by ELISA. VEGF levels are always significantly lower in supernatants from the cells that are seeded on Matrigel extracellular matrix (ECM) compared to the cells grown on plastic. Lower VEGF concentrations in supernatants from the pituitary cells cultured on ECM may reflect a more adequate cell environment compared to culture on plastic. These results demonstrate for the first time, that VEGF is expressed by lactotrophs, which outnumber FS cells. These results are of potential clinical relevance especially in oncology for the interpretation of studies investigating anti-angiogenic treatment of pituitary tumors.

Prolactin induces apoptosis of lactotropes in female rodents.

Anterior pituitary cell turnover occurring during female sexual cycle is a poorly understood process that involves complex regulation of cell proliferation and apoptosis by multiple hormones. In rats, the prolactin (PRL) surge that occurs at proestrus coincides with the highest apoptotic rate. Since anterior pituitary cells express the prolactin receptor (PRLR), we aimed to address the actual role of PRL in the regulation of pituitary cell turnover in cycling females. We showed that acute hyperprolactinemia induced in ovariectomized rats using PRL injection or dopamine antagonist treatment rapidly increased apoptosis and decreased proliferation specifically of PRL producing cells (lactotropes), suggesting a direct regulation of these cell responses by PRL. To demonstrate that apoptosis naturally occurring at proestrus was regulated by transient elevation of endogenous PRL levels, we used PRLR-deficient female mice (PRLRKO) in which PRL signaling is totally abolished. According to our hypothesis, no increase in lactotrope apoptotic rate was observed at proestrus, which likely contributes to pituitary tumorigenesis observed in these animals. To decipher the molecular mechanisms underlying PRL effects, we explored the isoform-specific pattern of PRLR expression in cycling wild type females. This analysis revealed dramatic changes of long versus short PRLR ratio during the estrous cycle, which is particularly relevant since these isoforms exhibit distinct signaling properties. This pattern was markedly altered in a model of chronic PRLR signaling blockade involving transgenic mice expressing a pure PRLR antagonist (TGΔ1–9-G129R-hPRL), providing evidence that PRL regulates the expression of its own receptor in an isoform-specific manner. Taken together, these results demonstrate that i) the PRL surge occurring during proestrus is a major proapoptotic signal for lactotropes, and ii) partial or total deficiencies in PRLR signaling in the anterior pituitary may result in pituitary hyperplasia and eventual prolactinoma development, as observed in TGΔ1–9-G129R-hPRL and PRLRKO mice, respectively.

Secondary amenorrhea in a woman with spinocerebellar degeneration treated with thyrotropin-releasing hormone: a case report and in vitro analysis

IntroductionWhile thyrotropin-releasing hormone is known to be a prolactin-release stimulating factor, thyrotropin-releasing hormone-tartrate and its derivative, taltirelin hydrate, are used for the treatment of spinocerebellar degeneration, a degenerative disease characterized mainly by motor ataxia. We report the case of a patient being treated with a thyrotropin-releasing hormone preparation for spinocerebellar degeneration who developed amenorrhea after a dose increase. Her hormonal background was analyzed and the effect of prolonged exposure to thyrotropin-releasing hormone on pituitary prolactin-producing cells was examined in vitro.Case presentationOur patient was a 36-year-old Japanese woman who experienced worsening of gait disturbance at around 23 years of age, and was subsequently diagnosed as having spinocerebellar degeneration. She had been treated with thyrotropin-releasing hormone-tartrate for four years. Taltirelin hydrate was added to the treatment seven months prior to her presentation, followed by an improvement in gait disturbance. Around the same period, she started lactating and subsequently developed amenorrhea three months later. Taltirelin hydrate was discontinued and she was referred to our hospital. She was found to have normal sex hormone levels. A thyrotropin-releasing hormone provocation test showed a normal response of thyroid-stimulating hormone level and an over-response of prolactin at 30 minutes (142.7 ng/mL). Resumption of menstruation was noted three months after dose reduction of thyrotropin-releasing hormone. In our in vitro study, following long-term exposure to thyrotropin-releasing hormone, cells from the rat pituitary prolactin-producing cell line GH3 exhibited an increased basal prolactin promoter activity but showed a marked decrease in responsiveness to thyrotropin-releasing hormone.ConclusionsPhysicians should be aware of hyperprolactinemia-associated side effects in patients receiving thyrotropin-releasing hormone treatment. Long-term treatment with a thyrotropin-releasing hormone preparation might cause a large amount of prolactin to accumulate in prolactin-producing cells and be released in response to exogenous thyrotropin-releasing hormone stimulation.

Stimulatory effect of pituitary adenylate-cyclase activating polypeptide (PACAP) and its PACAP type I receptor (PAC1R) on prolactin synthesis in rat pituitary somatolactotroph GH3 cells

In this present study, we investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor, PACAP type I receptor (PAC1R) on prolactin synthesis in pituitary somatolactotroph GH3 cells. PACAP increased prolactin promoter activity up to 1.3±0.1-fold. This increase, while significant, was less than the increase resulting from thyrotropin-releasing hormone (TRH) stimulation. By transfection of a PAC1R expression vector to the cells, the response to PACAP on prolactin promoter activity was dramatically potentiated to a degree proportional to the amount of PAC1R transfected. In the PAC1R expressing GH3 cells, TRH and PACAP alone increased prolactin promoter up to 3.3±0.3-fold and 4.9±0.2-fold, respectively, and combined treatment with TRH and PACAP further increased prolactin promoters up to 6.8±0.6-fold. PACAP binds both Gs- and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/extracellular signal-regulated kinase (ERK) signaling pathways. PACAP increased ERK phosphorylation in PAC1R expressing cells to the same degree as TRH. Combined treatment with TRH and PACAP had a synergistic effect on ERK activation. GH3 cells produce both prolactin and growth hormone. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin and attenuated growth hormone mRNA expression. PACAP increased both prolactin and growth hormone mRNA levels, particularly in PAC1R expressing cells. In addition, increasing amount of PAC1R in GH3 cells potentiated the action of TRH on prolactin promoter activity, as well as on ERK phosphorylation. PAC1R was induced by PACAP itself, but not by TRH. Our current study demonstrates that PACAP and its PAC1R, functions as a stimulator of prolactin alone or with TRH in prolactin producing cells.