Regulation of Pituitary Progenitor Differentiation by ß-Catenin.

The pituitary gland is a critical organ that is necessary for many physiological processes, including growth, reproduction, and stress response. The secretion of pituitary hormones from specific cell types regulates these essential processes. Pituitary hormone cell types arise from a common pool of pituitary progenitors, and mutations that disrupt the formation and differentiation of pituitary progenitors result in hypopituitarism. Canonical WNT signaling through CTNNB1 (ß-catenin) is known to regulate the formation of the POU1F1 lineage of pituitary cell types. When ß-catenin is deleted during the initial formation of the pituitary progenitors, Pou1f1 is not transcribed, which leads to the loss of the POU1F1 lineage. However, when ß-catenin is deleted after lineage specification, there is no observable effect. Similarly, the generation of a ß-catenin gain-of-function allele in early pituitary progenitors or stem cells results in the formation of craniopharyngiomas, whereas stimulating ß-catenin in differentiated cell types has no effect. PROP1 is a pituitary-specific transcription factor, and the peak of PROP1 expression coincides with a critical time point in pituitary organogenesis-that is, after pituitary progenitor formation but before lineage specification. We used a Prop1-cre to conduct both loss- and gain-of-function studies on ß-catenin during this critical time point. Our results demonstrate that pituitary progenitors remain sensitive to both loss and gain of ß-catenin at this time point, and that either manipulation results in hypopituitarism.

Premature Expression of FOXO1 in Developing Mouse Pituitary Results in Anterior Lobe Hypoplasia.

The process by which the somatotrope lineage emerges in the developing pituitary is regulated by the activity of specific signaling and transcription factors expressed during development. We set out to understand the contribution of FOXO1 to that process by using a mouse model in which FOXO1 is prematurely expressed in the pituitary primordium. Expression of FOXO1 in the oral ectoderm as early as embryonic day (e)9.5 resulted in pituitary gland hypoplasia and reduced expression of anterior lobe hormone transcripts at e18.5. Of note, the relative numbers of somatotropes and thyrotropes were also decreased at e18.5. LHX3 and PITX2, markers of pituitary identity, were present in a reduced number of cells during the formation of the Rathke pouch. Thus, premature expression of FOXO1 may affect adoption of pituitary identity during differentiation. Our results demonstrate that the timing of FOXO1 activation affects its role in pituitary gland organogenesis and somatotrope differentiation.

Low Thyroid Hormone Levels Disrupt Thyrotrope Development.

Low thyroid hormone (TH) conditions caused by a variety of prenatal and perinatal problems have been shown to alter postnatal regulatory thyrotropin (TSH) responsiveness to TH in humans and rodents. The mechanisms underlying this pituitary TH resistance remain unknown. Here we use the evolutionarily conserved zebrafish model to examine the effects of low TH on thyrotrope development and function. Zebrafish were exposed to the goitrogen 6-propyl-2-thiouracil (PTU) to block TH synthesis, and this led to an approximately 50% increase in thyrotrope numbers and an 8- to 10-fold increase in tshb mRNA abundance in 2-week-old larvae and 1-month-old juveniles. Thyrotrope numbers returned to normal 3 weeks after cessation of PTU treatment, demonstrating that these effects were reversible and revealing substantial plasticity in pituitary-thyroid axis regulation. Using a T4 challenge assay, we found that development under low-TH conditions did not affect the ability of T4 to suppress tshb mRNA levels despite the thyrotrope hyperplasia that resulted from temporary low-TH conditions. Together, these studies show that low developmental TH levels can lead to changes in thyrotrope number and function, providing a possible cellular mechanism underlying elevated TSH levels seen in neonates with either permanent or transient congenital hypothyroidism.

Derivation of Diverse Hormone-Releasing Pituitary Cells from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) provide an unlimited cell source for regenerative medicine. Hormone-producing cells are particularly suitable for cell therapy, and hypopituitarism, a defect in pituitary gland function, represents a promising therapeutic target. Previous studies have derived pituitary lineages from mouse and human ESCs using 3D organoid cultures that mimic the complex events underlying pituitary gland development in vivo. Instead of relying on unknown cellular signals, we present a simple and efficient strategy to derive human pituitary lineages from hPSCs using monolayer culture conditions suitable for cell manufacturing. We demonstrate that purified placode cells can be directed into pituitary fates using defined signals. hPSC-derived pituitary cells show basal and stimulus-induced hormone release in vitro and engraftment and hormone release in vivo after transplantation into a murine model of hypopituitarism. This work lays the foundation for future cell therapy applications in patients with hypopituitarism.

The CpG island in the murine foxl2 proximal promoter is differentially methylated in primary and immortalized cells.

Forkhead box L2 (Foxl2), a member of the forkhead transcription factor family, plays important roles in pituitary follicle-stimulating hormone synthesis and in ovarian maintenance and function. Mutations in the human FOXL2 gene cause eyelid malformations and premature ovarian failure. FOXL2/Foxl2 is expressed in pituitary gonadotrope and thyrotrope cells, the perioptic mesenchyme of the developing eyelid, and ovarian granulosa cells. The mechanisms governing this cell-restricted expression have not been described. We mapped the Foxl2 transcriptional start site in immortalized murine gonadotrope-like cells, LßT2, by 5' rapid amplification of cDNA ends and then PCR amplified approximately 1 kb of 5' flanking sequence from murine genomic DNA. When ligated into a reporter plasmid, the proximal promoter conferred luciferase activity in both homologous (LßT2) and, unexpectedly, heterologous (NIH3T3) cells. In silico analyses identified a CpG island in the proximal promoter and 5' untranslated region, suggesting that Foxl2 transcription might be regulated epigenetically. Indeed, pyrosequencing and quantitative analysis of DNA methylation using real-time PCR revealed Foxl2 proximal promoter hypomethylation in homologous compared to some, though not all, heterologous cell lines. The promoter was also hypomethylated in purified murine gonadotropes. In vitro promoter methylation completely silenced reporter activity in heterologous and homologous cells. Collectively, the data suggest that differential proximal promoter DNA methylation may contribute to cell-specific Foxl2 expression in some cellular contexts. However, gonadotrope-specific expression of the gene cannot be explained by promoter hypomethylation alone.

Triiodothyronine rapidly alters the TSH content and the secretory granules distribution in male rat thyrotrophs by a cytoskeleton rearrangement-independent mechanism.

Rapid actions of T3 on TSH synthesis in posttranscriptional steps, such as polyadenylation and translation rate, have already been described. The focus of this paper was to characterize rapid actions of T3 on TSH secretion and the involvement of actin and microtubule cytoskeleton in this process. For that, sham-operated (SO) and thyroidectomized (Tx) rats were subjected to acute or chronic treatment with T3. We observed a disarrangement in microtubule and actin cytoskeletons and an increase in Tshb mRNA levels in Tx rats, whereas the total TSH protein content was reduced in the pituitary gland as a whole, but increased in the secretory granules close to the plasma membrane of thyrotrophs, as well as in the extracellular space. The acute T3 dose promoted a rapid increase and redistribution of TSH secretory granules throughout the cytoplasm, as well as a rearrangement in actin and microtubule cytoskeletons. The T3 chronic treatment outcome reinforces the acute effects observed and, additionally, evinces an increase in the α-tubulin content and a rearrangement in microtubule cytoskeleton. Thus, T3 is able to rapidly suppress TSH secretion and, in parallel, to promote a rearrangement in actin and microtubules assembly throughout the pituitary gland, effects that seem to be independent from each other.

PPARα modulates the TSH ß-subunit mRNA expression in thyrotrope TαT1 cells and in a mouse model.

SCOPE: Fasting leads to a significant downregulation of the hypothalamus-pituitary-thyroid axis, and peroxisome proliferator-activated receptor (PPAR) α is a key transcription factor in mediating a magnitude of adaptive responses to fasting. In this study, we examined the role of PPARα in regulation of the hypothalamus-pituitary-thyroid axis. METHODS AND RESULTS: Thyroid-stimulating hormone ß-subunit (TSHß) mRNA abundance was being reduced in response to treatment of TαT1 cells with PPARα agonists (p < 0.05), indicating an inhibitory transcriptional regulation of TSHß by PPARα. As expected, fasting significantly downregulated TSHß mRNA expression in a two-factorial study with fed or fasted wild-type (WT) and PPARα knockout mice (p < 0.05). In contrast to the in vitro data, fasted PPARα knockout mice revealed lower mRNA concentrations of pituitary TSHß (-64%) and TSH-regulated thyroid genes, and lower plasma concentrations of thyroxine (T4, -25%), triiodothyronine (T3, -25%), free T4 (-60%), and free T3 (-35%) than fasted WT mice (p < 0.05). Those differences were not observed in fed mice. CONCLUSIONS: Data from thyrotrope cells revealed that PPARα could contribute to the fasting-associated downregulation of the TSHß mRNA expression. In a mouse model, fasting led to a significant reduction in TSHß mRNA level, but unexpectedly this effect was stronger in mice lacking PPARα than in WT mice.

Thymulin gene therapy prevents the histomorphometric changes induced by thymulin deficiency in the thyrotrope population of mice.

There is evidence of the existence of a bidirectional relationship between the thymus gland and the thyroid axis. Since the thymic peptide thymulin possesses hypophysiotropic activity, we undertook the task of assessing the histomorphometric changes induced by thymulin deficiency on the thyrotrope population of normal mice and the action of neonatal thymulin gene therapy on the thyrotropin (TSH)-cells of nude mice. C57BL/6 mice were subjected to immunoneutralization of circulating thymulin from postnatal day 1 to the end of the study (postnatal day 32) by intraperitoneal injections of rabbit anti-factor thymulin serum (α-FTS) and normal rabbit serum in controls. Also, neonatal thymulin gene therapy was implemented in athymic nude mice using an adenoviral vector expressing a gene for thymulin (RAd-FTS). On postnatal day 1, heterozygous (nu/+) and homozygous (nu/nu) pups received a single bilateral intramuscular (i.m.) injection of either RAd-FTS or RAd-GFP (the latter being the control vector). The pituitaries were immunostained for TSH. Thymulin immunoneutralization severely reduced serum thymulin (p < 0.01). We detected a significant (p < 0.05) decrease in cell size (CS) and volume density (VD) with a nonsignificant decrease in cell density (CD) in C57BL/6 in both males and females. A single neonatal i.m. injection of RAd-FTS markedly increased the circulating levels of serum thymulin in the athymic mice and increased the CD (p < 0.05), CS (p < 0.01) and VD (p < 0.01) of the thyrotrope population in nu/nu mice. Thyroid histology was not affected. Our results suggest a possible modulating effect of thymulin on the thyrotrope population.

Suppressive effects of genistein and daidzein on pituitary-thyroid axis in orchidectomized middle-aged rats.

High intake of soybean phytoestrogens, isoflavones genistein (G) and daidzein (D), has been associated with health benefits. However, isoflavones were reported to affect adversely thyroid function in the presence of other goitrogenic factors. As the thyroid gland becomes functionally impaired with age, we examined whether supplementary doses of G or D would affect morphology and function of pituitary-thyroid axis in middle-aged male rats. Sixteen-month-old orchidectomized Wistar rats were treated with 10 mg/kg of either G or D, while the control sham-operated and orchidectomized group received just the vehicle for three weeks. The animals were fed soy-free diet with increased iodine content, and killed 24 h after the last treatment. Their pituitaries and thyroids were excised and prepared for further immunohistochemical and morphometric investigation. The concentrations of thyroid-stimulating hormone (TSH), total T(4) and T(3), in the serum were determined. In both isoflavone-treated groups, pituitary TSH-immunopositive cells had increased cellular volume and relative volume density (P < 0.05), as well as increased serum TSH levels (P < 0.05) in comparison to the controls; their thyroid tissue was characterized by increased volume of thyroglobulin-immunopositive epithelium (P < 0.05), epithelial height and index of activation rate (P < 0.05), while the volume of luminal colloid, and total serum T(4) and T(3) levels decreased (P < 0.05) in comparison to the controls. In conclusion, this study provides the first direct evidence that both G and D can induce microfollicular changes in the thyroid tissue and reduce the level of thyroid hormones in Orx middle-aged male rats, a model of andropause. This reduction consequently led to a feedback stimulation of pituitary TSH cells. The detected stimulatory effect was higher in the daidzein-treated rats.

Therapeutic concentrations of mitotane (o,p'-DDD) inhibit thyrotroph cell viability and TSH expression and secretion in a mouse cell line model.

Mitotane therapy is associated with many side effects, including thyroid function perturbations mimicking central hypothyroidism, possibly due to laboratory test interference or pituitary direct effects of mitotane. We investigated whether increasing concentrations of mitotane in the therapeutic range might interfere with thyroid hormone assays and evaluated the effects of mitotane on a mouse TSH-producing pituitary cell line. TSH, free T(4), and free T(3) levels do not significantly change in sera from hypo-, hyper-, or euthyroid patients after addition of mitotane at concentrations in the therapeutic window. In the mouse TalphaT1 cell line, mitotane inhibits both TSH expression and secretion, blocks TSH response to TRH, and reduces cell viability, inducing apoptosis at concentrations in the therapeutic window. TRH is not capable of rescuing TalphaT1 cells from the inhibitory effects of mitotane on TSH expression and secretion, which appear after short time treatment and persist over time. Our results demonstrate that mitotane does not interfere with thyroid hormone laboratory tests but directly reduces both secretory activity and cell viability on pituitary TSH-secreting mouse cells. These data represent a possible explanation of the biochemical picture consistent with central hypothyroidism in patients undergoing mitotane therapy and open new perspectives on the direct pituitary effects of this drug.

Unbiased stereological estimation of the rat fetal pituitary volume and of the total number and volume of TSH cells after maternal dexamethasone application.

Glucocorticoids have an inhibitory influence on proliferation activity of the pituitary cells while stimulating apoptosis. Therefore, it was hypothesized that the synthetic glucocorticoid, dexamethasone (DX), has an inhibitory influence on the number of thyroid-stimulating hormone (TSH) cells during fetal development. The effects of maternal administration of DX on stereological parameters of TSH cells, and TSH serum concentration were investigated in 21-day-old rat fetuses. On day 16 of pregnancy, the experimental dams received 1.0 mg DX/kg b.w. subcutaneously, followed by 0.5 mg DX/kg b.w./day on days 17 and 18 of gestation. The control gravid females received the same volume of saline vehicle. TSH cells were stained immunocytochemically by the peroxidase-antiperoxidase (PAP) method. The fetal pituitary volumes were estimated using Cavalieri's principle. A physical disector counting technique in combination with the fractionator sampling method was used for estimation of pituitary TSH cell number. Cell and nuclear volumes were measured with a planar rotator. Maternal DX application was found to cause a significant decrease of pituitary volume and number of TSH cells per pituitary in 21-day-old fetuses in comparison with the control fetuses. TSH cell number expressed per body weight unit declined significantly after maternal DX administration. These results indicate an inhibitory DX influence on proliferative activity of precursors and likely differentiated TSH cells and increased apoptotic prevalence. The histological appearance, volume of TSH cells and TSH serum concentration suggest intensive synthetic activity in TSH cells of DX exposed fetuses.

Expression patterns of hormones, signaling molecules, and transcription factors during adenohypophysis development in the chick embryo.

The chick embryo is an ideal model to study pituitary cell-type differentiation. Previous studies describing the temporal appearance of differentiated pituitary cell types in the chick embryo are contradictory. To resolve these controversies, we used RT-PCR to define the temporal onset and in situ hybridization and immunohistochemistry to define the spatial localization of hormone expression within the pituitary. RT-PCR detected low levels of Fshbeta (gonadotropes) and Pomc (corticotropes, melanotropes) mRNA at E4 and Gh (somatotropes), Prl (lactotropes), and Tshbeta (thyrotropes) mRNA at E8. For all hormones, sufficient accumulation of mRNA and/or protein to permit detection by in situ hybridization or immunohistochemistry was observed approximately 3 days later and in all cases corresponded to a notable increase in RT-PCR product. We also describe the expression patterns of signaling (Bmp2, Bmp4, Fgf8, Fgf10, Shh) and transcription factors (Pitx1, Pitx2, cLim3) known to be important for pituitary organogenesis in other model organisms.

Immunohistochemical and morphometric study of pituitary pars distalis thyrotrophs of male viscacha (Lagostomus maximus maximus): seasonal variations and effect of melatonin and castration.

The physiology and behavior of viscacha vary along the year according to the modifications of environmental signals such as the photoperiod length, temperature, rainfall pattern, food composition, and social interactions. The pituitary pars distalis thyrotrophs (TSH cells) of male viscacha were immunohistochemically identified, and the morphometric parameters: percentage of immunopositive area (% IA), cell percentage in pars distalis (% PDC), number of cells per reference area (no. cells/RA), and major cellular and nuclear diameters were analyzed. Three different groups of adult male viscachas were used: (1) captured in their natural habitat during the year, (2) melatonin-administered, and (3) castrated. The thyrotrophs were localized in the ventromedial sector, mainly in the pars distalis cephalic extreme. They were oval or pyramidal in shape, and their immunostaining intensity was heterogeneous. The % IA, % PDC, and no. cells/RA exhibited a significant decrease in June-July (winter, gonadal regression period) in relation to February-March (summer-early autumn, reproductive period), and they were recovered in August-September (later winter-early spring, gonadal recovery period). No morphometric variations of TSH cells were observed in melatonin-treated animals, whereas a decrease of the % IA, % PDC, and no. cells/RA was observed in castrated animals in relation to the intact animals. Our results show TSH cell morphometric variations during the year in agreement with the animal's different physiological conditions during the reproductive cycle, and probably in response to the environmental signals changes. Melatonin does not have a direct effect on the TSH cells. However, castration modifies some thyrotroph morphometric parameters, reinforcing the hypothesis that androgens affect the cells activity.

Hypothalamic input is required for development of normal numbers of thyrotrophs and gonadotrophs, but not other anterior pituitary cells in late gestation sheep.

To evaluate the hypothalamic contribution to the development of anterior pituitary (AP) cells we surgically disconnected the hypothalamus from the pituitary (hypothalamo-pituitary disconnection, HPD) in fetal sheep and collected pituitaries 31 days later. Pituitaries (n = 6 per group) were obtained from fetal sheep (term = 147 +/- 3 days) at 110 days (unoperated group) of gestation and at 141 days from animals that had undergone HPD or sham surgery at 110 days. Cells were identified by labelling pituitary sections with antisera against the six AP hormones. Additionally, we investigated the colocalization of glycoprotein hormones. The proportions of somatotrophs and corticotrophs were unchanged by age or HPD. Lactotrophs increased 80% over time, but the proportion was unaffected by HPD. Thyrotrophs, which were unaffected by age, increased 70% following HPD. Gonadotrophs increased with gestational age (LH+ cells 55%; FSH+ cells 19-fold), but this was severely attenuated by HPD. We investigated the possible existence of a reciprocal effect of HPD on multipotential glycoprotein-expressing cells. Co-expression of LH and TSH was extremely rare (< 1%) and unchanged over the last month of gestation or HPD. The increase of gonadotrophs expressing FSH only or LH and FSH was attenuated by HPD. Therefore, the proportions of somatotrophs, lactotrophs and corticotrophs are regulated independently of hypothalamic input in the late gestation fetal pituitary. In marked contrast, the determination of the thyrotroph and gonadotroph lineages over the same time period is subject to complex mechanisms involving hypothalamic factors, which inhibit differentiation and/or proliferation of thyrotrophs, but stimulate gonadotrophs down the FSH lineage. Development of a distinct population of gonadotrophs, expressing only LH, appears to be subject to alternative mechanisms.

Hes1 is required for pituitary growth and melanotrope specification.

Rathke's pouch contains progenitor cells that differentiate into the endocrine cells of the pituitary gland. It gives rise to gonadotrope, thyrotrope, somatotrope, corticotrope and lactotrope cells in the anterior lobe and the intermediate lobe melanotropes. Pituitary precursor cells express many members of the Notch signaling pathway including the downstream effector gene Hes1. We hypothesized that Hes1 regulates the timing of precursor differentiation and cell fate determination. To test this idea, we expressed Hes1 in differentiating pituitary cells and found that it can inhibit gonadotrope and thyrotrope differentiation. Pituitaries of Hes1 deficient mice have anterior lobe hypoplasia. All cells in the anterior lobe are specified and differentiate, but an early period of increased cell death and reduced proliferation causes reduced growth, evident as early as e14.5. In addition, cells within the intermediate lobe differentiate into somatotropes instead of melanotropes. Thus, the Hes1 repressor is essential for melanotrope specification. These results demonstrate that Notch signaling plays multiple roles in pituitary development, influencing precursor number, organ size, cell differentiation and ultimately cell fate.

Morphometric assessment of the impact of serum thymulin immunoneutralization on pituitary cell populations in peripubertal mice.

Thymulin is a thymic hormone involved in several aspects of intra- and extrathymic T-cell differentiation. Thymulin also possesses hypophysiotropic activity which suggests that this metallopeptide may play an important role in thymus-pituitary communication, particularly during early life. The aim of the present study was to evaluate the impact of serum thymulin suppression from birth to peripuberty on the morphology of different pituitary cell populations in prepubertal C57Bl/6 mice. Animals were submitted to immunoneutralization of circulating thymulin from postnatal day 1 to the end of the study (age 32 days). From their 1st day of life, the animals were submitted to a protocol of intraperitoneal injections of rabbit anti-thymulin serum (alpha-FTS) and normal rabbit serum (NRS) in the controls. On their 33rd day of life, the animals were killed and their pituitaries were immediately dissected, fixed and immunostained using the EnVision system with primary antibodies against growth hormone, thyrotropin, corticotropin, gonadotropins and prolactin. Morphometry was performed by means of an image analysis system. The following parameters were calculated: volume density = Sigma cell area/reference area (RA); cell density (CD) = number of cells/RA, and cell size (expressed in microm2). Serum thymulin was measured by a rosette bioassay while pituitary hormones were assayed by radioimmunoassay. Serum prolactin, luteinizing hormone, follicle-stimulating hormone, growth hormone and thyroid-stimulating hormone were significantly lower in the alpha-FTS animals of either sex compared with the corresponding NRS counterparts. The somatotrope, lactotrope and corticotrope populations showed a significant decrease in CD, while cell hypertrophy was observed in some of the pituitary cell populations of the alpha-FTS group compared to the NRS group. In the alpha-FTS group, there were sex differences in the morphometric changes observed. Our results suggest that serum thymulin plays a significant role during early life in the postnatal maturation of endocrine cells of the mouse anterior pituitary gland.

A histochemical investigation of different distribution of TSH cells in adenohypophsis of female and male rats.

The aim of the present study was to investigate histochemically the localization of thyroid-stimulating hormone (TSH) cells and the number of TSH cells in adenohypophysis of ovariectomized adult females, intact females at estrous and diestrous phase of sexual cycles and castrated and non-castrated adult male rats. TSH cells, distributed throughout the pars distalis in all groups, were observed more frequent in areas near to the pars intermedia than other regions. The cells were round, ovoid, stellar-shaped and had contacts with sinusoids. Their nuclei were large and round-shaped. The cells were found mostly single or occasionally in groups of double and triple. The number of TSH cells was higher in estrous than diestrous phase in intact rats. In adenohypophysis of ovariectomized rats, their numbers were less than that of intacts in estrous, but higher than that of found in diestrous phases. Furthermore, the numbers of TSH cells in intact females during estrous phase was higher than those found in intact and castrated male rats. On the other hand, castration reduced the number of TSH cells while ovariectomy had no effect in this respect.