The effects of short-term and long-term phthalate exposures on ovarian follicle growth dynamics and hormone levels in female mice†.

Di(2-ethylhexyl) phthalate and diisononyl phthalate are widely used as plasticizers in polyvinyl chloride products. Short-term exposures to phthalates affect hormone levels, ovarian follicle populations, and ovarian gene expression. However, limited data exist regarding the effects of long-term exposure to phthalates on reproductive functions. Thus, this study tested the hypothesis that short-term and long-term exposure to di(2-ethylhexyl) phthalate or diisononyl phthalate disrupts follicle dynamics, ovarian and pituitary gene expression, and hormone levels in female mice. Adult CD-1 female mice were exposed to vehicle, di(2-ethylhexyl) phthalate, or diisononyl phthalate (0.15 ppm, 1.5 ppm, or 1500 ppm) via the chow for 1 or 6 months. Short-term exposure to di(2-ethylhexyl) phthalate (0.15 ppm) and diisononyl phthalate (1.5 ppm) decreased serum follicle-stimulating hormone levels compared to control. Long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate (1500 ppm) increased the percentage of primordial follicles and decreased the percentages of preantral and antral follicles compared to control. Both phthalates increased follicle-stimulating hormone levels (di(2-ethylhexyl) phthalate at 1500 ppm; diisononyl phthalate at 1.5 ppm) and decreased luteinizing hormone levels (di(2-ethylhexyl) phthalate at 0.15 and 1.5 ppm; diisononyl phthalate at 1.5 ppm and 1500 ppm) compared to control. Furthermore, both phthalates altered the expression of pituitary gonadotropin subunit genes (Cga, Fshb, and Lhb) and a transcription factor (Nr5a1) that regulates gonadotropin synthesis. These data indicate that long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate alters follicle growth dynamics in the ovary and the expression of gonadotropin subunit genes in the pituitary and consequently luteinizing hormone and follicle-stimulating hormone synthesis.

Dietary exposure to an environmentally relevant phthalate mixture alters follicle dynamics, hormone levels, ovarian gene expression, and pituitary gene expression in female mice.

Phthalates are chemicals ubiquitously used in industry. Individual phthalates have been found to adversely affect female reproduction; however, humans are exposed to a mixture of phthalates daily, primarily through ingestion. Previous studies show that exposure to an environmentally relevant mixture of phthalates (Mix) can affect female reproduction. Little research, however, has been conducted on the effects of short-term (1 month) and long-term (6 months) exposure to Mix on ovarian functions. Thus, this study tested the hypothesis that short-term and long-term exposure to Mix alters ovarian folliculogenesis, serum hormone concentrations, pituitary gene expression, and ovarian expression of genes involved in steroidogenesis, apoptosis, cell cycle regulation, and oxidative stress. Adult CD-1 female mice were exposed to vehicle control (corn oil) or Mix (0.15-1500 ppm) in the chow for 1 or 6 months. Exposure to Mix for 1 month increased the number of atretic follicles (0.15 ppm), altered ovarian gene expression (0.15 ppm, 1500 ppm), and decreased serum testosterone (1.5 ppm) compared to control. Exposure to Mix for 6 months increased serum follicle-stimulating hormone (FSH) (0.15 ppm), decreased serum luteinizing hormone (LH) (0.15 ppm, 1.5 ppm, and 1500 ppm), decreased serum estradiol (1500 ppm), altered pituitary gene expression (1500 ppm), increased the number (1500 ppm) and percentage (1.5 ppm and 1500 ppm) of primordial follicles, and decreased the percentage of preantral (1500 ppm) and antral (1.5 ppm and 1500 ppm) follicles compared to control. These data indicate that exposure to Mix can alter folliculogenesis, steroidogenesis, and gene expression in female mice.

Characterization of Somatotrope Cell Expansion in Response to GHRH in the Neonatal Mouse Pituitary.

In humans and mice, loss-of-function mutations in growth hormone-releasing hormone receptor (GHRHR) cause isolated GH deficiency. The mutant GHRHR mouse model, GhrhrLit/Lit (LIT), exhibits loss of serum GH, but also fewer somatotropes. However, how loss of GHRH signaling affects expansion of stem and progenitor cells giving rise to GH-producing cells is unknown. LIT mice and wild-type littermates were examined for differences in proliferation and gene expression of pituitary lineage markers by quantitative reverse transcription polymerase chain reaction and immunohistochemistry at postnatal day 5 (p5) and 5 weeks. At p5, the LIT mouse shows a global decrease in pituitary proliferation measured by proliferation marker Ki67 and phospho-histone H3. This proliferative defect is seen in a pituitary cell expressing POU1F1 with or without GH. SOX9-positive progenitors show no changes in proliferation in p5 LIT mice. Additionally, the other POU1F1 lineage cells are not decreased in number; rather, we observe an increase in lactotrope cell population as well as messenger RNA for Tshb and Prl. In the 5-week LIT pituitary, the proliferative deficit in POU1F1-expressing cells observed neonatally persists, while the number and proliferative proportion of SOX9 cells do not appear changed. Treatment of cultured pituitary explants with GHRH promotes proliferation of POU1F1-expressing cells, but not GH-positive cells, in a mitogen-activated protein kinase-dependent manner. These findings indicate that hypothalamic GHRH targets proliferation of a POU1F1-positive cell, targeted to the somatotrope lineage, to fine tune their numbers.

Prenatal and postnatal exposure to polychlorinated biphenyls alter follicle numbers, gene expression, and a proliferation marker in the rat ovary.

Polychlorinated biphenyls (PCBs) were used in industrial applications until they were banned in the 1970s, but they still persist in the environment. Little is known about the long-term effects of exposure to PCB mixtures on the rat ovary during critical developmental periods. Thus, this study tested whether prenatal and postnatal exposures to PCBs affect follicle numbers and gene expression in the ovaries of F1 offspring. Sprague-Dawley rats were treated with vehicle or Aroclor 1221 (A1221) at 1 mg/kg/day during embryonic days 8-18 and/or postnatal days (PND) 1-21. Ovaries from F1 rats were collected for assessment of follicle numbers and differential expression of estrogen receptor 1 (Esr1), estrogen receptor 2 (Esr2), androgen receptor (Ar), progesterone receptor (Pgr), and Ki-67 (Ki67) at PNDs 8, 32, and 60. Sera were collected for measurement of estradiol concentrations. Prenatal exposure to A1221 significantly decreased the number of primordial follicles and the total number of follicles at PND 32 compared to control. Postnatal PCB exposure borderline increased Ki67 gene expression and significantly increased Ki67 protein levels (PND 60) compared to control. Combined prenatal and postnatal PCB exposure borderline decreased Ar expression (PND 8) compared to control. However, PCB exposure did not significantly affect the expression of Pgr, Esr1, and Esr2 or serum estradiol concentrations compared to control at any time point. In conclusion, these data suggest that PCB exposure affects follicle numbers and levels of the proliferation marker Ki67, but it does not affect expression of some sex steroid hormone receptors in the rat ovary.

Pre- and postnatal developmental exposure to the polychlorinated biphenyl mixture aroclor 1221 alters female rat pituitary gonadotropins and estrogen receptor alpha levels.

Polychlorinated-biphenyls (PCBs) are industrial compounds, which were widely used in manufacturing of electrical parts and transformers. Despite being banned in 1979 due to human health concerns, they persist in the environment. In humans and experimental model systems, PCBs elicit toxicity in part by acting as endocrine-disrupting chemicals (EDCs). Aroclor 1221 (A1221) is a weakly estrogenic PCB mixture known to alter reproductive function in rodents. EDCs can impact hormone signaling at any level of the hypothalamic-pituitary-gonadal (HPG) axis, and we investigated the effects of A1221 exposure during the prenatal and postnatal developmental periods on pituitary hormone and steroid receptor expression in female rats. Examining offspring at 3 ages, postnatal day 8 (P8), P32 and P60, we found that prenatal exposure to A1221 increased P8 neonate pituitary luteinizing hormone beta (Lhb) mRNA and LHß gonadotrope cell number while decreasing LH serum hormone concentration. No changes in pituitary hormone or hormone receptor gene expression were observed peri-puberty at P32. In reproductively mature rats at P60, we found pituitary follicle stimulating hormone beta (Fshb) mRNA levels increased by prenatal A1221 exposure with no corresponding alterations in FSH hormone or FSHß expressing cell number. Estrogen receptor alpha (ERα) mRNA and protein levels were also increased at P60, but only following postnatal A1221 dosing. Together, these data illustrate that exposure to the PCB A1221, during critical developmental windows, alters pituitary gonadotropin hormone subunits and ERα levels in offspring at different phases of maturation, potentially impacting reproductive function in concert with other components of the HPG axis.

Female-specific pituitary gonadotrope dysregulation in mice with chronic focal epilepsy.

Gonadotropin hormone release from the anterior pituitary is critical to regulating reproductive endocrine function. Clinical evidence has documented that people with epilepsy display altered levels of gonadotropin hormones, both acutely following seizures and chronically. Despite this relationship, pituitary function remains a largely understudied avenue in preclinical epilepsy research. Recently, we showed that females in the intrahippocampal kainic acid (IHKA) mouse model of temporal lobe epilepsy displayed changes in pituitary expression of gonadotropin hormone and gonadotropin-releasing hormone (GnRH) receptor genes. Circulating gonadotropin hormone levels, however, have yet to be measured in an animal model of epilepsy. Here, we evaluated the circulating levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), GnRH receptor (Gnrhr) gene expression, and sensitivity to exogenous GnRH in IHKA males and females. Although no changes in overall dynamics of pulsatile patterns of LH release were found in IHKA mice of either sex, estrus vs. diestrus changes in basal and mean LH levels were larger in IHKA females with prolonged, disrupted estrous cycles. In addition, IHKA females displayed increased pituitary sensitivity to GnRH and higher Gnrhr expression. The hypersensitivity to GnRH was observed on diestrus, but not estrus. Chronic seizure severity was not found to be correlated with LH parameters, and FSH levels were unchanged in IHKA mice. These results indicate that although there are changes in pituitary gene expression and sensitivity to GnRH in IHKA females, there may also be compensatory mechanisms that aid in maintaining gonadotropin release in the state of chronic epilepsy in this model.

Phenotypic differences based on lateralization of intrahippocampal kainic acid injection in female mice.

Clinical evidence indicates that patients with temporal lobe epilepsy (TLE) often show differential outcomes of comorbid conditions in relation to the lateralization of the seizure focus. A particularly strong relationship exists between the side of seizure focus and the propensity for distinct reproductive endocrine comorbidities in women with TLE. Therefore, here we evaluated whether targeting of left or right dorsal hippocampus for intrahippocampal kainic acid (IHKA) injection, a model of TLE, produces different outcomes in hippocampal granule cell dispersion, body weight gain, and multiple measures of reproductive endocrine dysfunction in female mice. One, two, and four months after IHKA or saline injection, in vivo measurements of estrous cycles and weight were followed by ex vivo examination of hippocampal dentate granule cell dispersion, circulating ovarian hormone and corticosterone levels, ovarian morphology, and pituitary gene expression. IHKA mice with right-targeted injection (IHKA-R) showed greater granule cell dispersion and pituitary Fshb expression compared to mice with left-targeted injection (IHKA-L). By contrast, pituitary expression of Lhb and Gnrhr were higher in IHKA-L mice compared to IHKA-R, but these values were not different from respective saline-injected controls. IHKA-L mice also showed an increased rate of weight gain compared to IHKA-R mice. Increases in estrous cycle length, however, were similar in both IHKA-L and IHKA-R mice. These findings indicate that although major reproductive endocrine dysfunction phenotypes present similarly after targeting left or right dorsal hippocampus for IHKA injection, distinct underlying mechanisms based on lateralization of epileptogenic insult may contribute to produce similar emergent reproductive endocrine outcomes.

Tributyltin and the Female Hypothalamic-Pituitary-Gonadal Disruption.

The hypothalamic-pituitary-gonadal (HPG) axis is the principal modulator of reproductive function. Proper control of this system relies on several hormonal pathways, which make the female reproductive components susceptible to disruption by endocrine-disrupting chemicals such as tributyltin (TBT). Here, we review the relevant research on the associations between TBT exposure and dysfunction of the female HPG axis components. Specifically, TBT reduced hypothalamic gonadotropin-releasing hormone (GnRH) expression and gonadotropin release, and impaired ovarian folliculogenesis, steroidogenesis, and ovulation, at least in part, by causing abnormal sensitivity to steroid feedback mechanisms and deleterious ovarian effects. This review covers studies using environmentally relevant doses of TBT in vitro (1 ng-20 ng/ml) and in vivo (10 ng-20 mg/kg) in mammals. The review also includes discussion of important gaps in the literature and suggests new avenue of research to evaluate the possible mechanisms underlying TBT-induced toxicity in the HPG axis. Overall, the evidence indicates that TBT exposure is associated with toxicity to the components of the female reproductive axis. Further studies are needed to better elucidate the mechanisms through which TBT impairs the ability of the HPG axis to control reproduction.

Iodoacetic Acid, a Water Disinfection Byproduct, Disrupts Hypothalamic, and Pituitary Reproductive Regulatory Factors and Induces Toxicity in the Female Pituitary.

Iodoacetic acid (IAA) is a water disinfection byproduct (DBP) formed by reactions between oxidizing disinfectants and iodide. In vitro studies have indicated that IAA is one of the most cyto- and genotoxic DBPs. In humans, DBPs have been epidemiologically associated with reproductive dysfunction. In mouse ovarian culture, IAA exposure significantly inhibits antral follicle growth and reduces estradiol production. Despite this evidence, little is known about the effects of IAA on the other components of the reproductive axis: the hypothalamus and pituitary. We tested the hypothesis that IAA disrupts expression of key neuroendocrine factors and directly induces cell damage in the mouse pituitary. We exposed adult female mice to IAA in drinking water in vivo and found 0.5 and 10 mg/l IAA concentrations lead to significantly increased mRNA levels of kisspeptin (Kiss1) in the arcuate nucleus although not affecting Kiss1 in the anteroventral periventricular nucleus. Both 10 mg/l IAA exposure in vivo and 20 µM IAA in vitro reduced follicle stimulating hormone (FSHß)-positive cell number and Fshb mRNA expression. IAA did not alter luteinizing hormone (LHß) expression in vivo although exposure to 20 µM IAA decreased expression of Lhb and glycoprotein hormones, alpha subunit (Cga) mRNA in vitro. IAA also had toxic effects in the pituitary, inducing DNA damage and P21/Cdkn1a expression in vitro (20 µM IAA) and DNA damage and Cdkn1a expression in vivo (500 mg/l). These data implicate IAA as a hypothalamic-pituitary-gonadal axis toxicant and suggest the pituitary is directly affected by IAA exposure.

Genistein inhibits proliferation and induces senescence in neonatal mouse pituitary gland explant cultures.

Genistein is an isoflavone abundant in soybean and infants are exposed to high levels of genistein in soy-based formula. It is known that genistein mediates estrogen receptor (ER) signaling, and exposure during neonatal development could cause acute and long term endocrine effects. We assayed genistein's impact on the neonatal mouse pituitary gland because it is an endocrine signaling hub and is sensitive to endocrine disruption during critical periods. Pituitary explant cultures, which actively proliferate and differentiate, were exposed to 0.06 µM-36 µM genistein and assayed for mRNA and protein changes. Genistein induced mRNA expression of the ERα regulated gene, Cckar, to the same magnitude as estradiol (E2) but with less potency. Interestingly, 36 µM genistein strongly inhibited pituitary proliferation, measured by a reduction in mKi67 mRNA and phospho-Histone H3 immunostaining. Examining cell cycle dynamics, we found that 36 µM genistein decreased Ccnb1 (Cyclin B1) mRNA; while mRNA for the cyclin dependent kinase inhibitor Cdkn1a (p21) was upregulated, correlated with an apparent increase in p21 immunostained cells. Strikingly, we observed a robust onset of cellular senescence, permanent cell cycle exit, in 36 µM genistein treated pituitaries by increased senescence activated ß-galactosidase staining. We also found that 36 µM genistein decreased Bcl2 mRNA levels, a gene protective against apoptosis. Taken together these data suggest that genistein exposure during the neonatal period could initiate senescence and halt proliferation during a time when the proper numbers of endocrine cells are being established for mature gland function.

Spatial patterning of liver progenitor cell differentiation mediated by cellular contractility and Notch signaling.

The progenitor cells of the developing liver can differentiate toward both hepatocyte and biliary cell fates. In addition to the established roles of TGFß and Notch signaling in this fate specification process, there is increasing evidence that liver progenitors are sensitive to mechanical cues. Here, we utilized microarrayed patterns to provide a controlled biochemical and biomechanical microenvironment for mouse liver progenitor cell differentiation. In these defined circular geometries, we observed biliary differentiation at the periphery and hepatocytic differentiation in the center. Parallel measurements obtained by traction force microscopy showed substantial stresses at the periphery, coincident with maximal biliary differentiation. We investigated the impact of downstream signaling, showing that peripheral biliary differentiation is dependent not only on Notch and TGFß but also E-cadherin, myosin-mediated cell contractility, and ERK. We have therefore identified distinct combinations of microenvironmental cues which guide fate specification of mouse liver progenitors toward both hepatocyte and biliary fates.

Cellular fate decisions in the developing female anteroventral periventricular nucleus are regulated by canonical Notch signaling.

The hypothalamic anteroventral periventricular nucleus (AVPV) is the major regulator of reproductive function within the hypothalamic-pituitary-gonadal (HPG) axis. Despite an understanding of the function of neuronal subtypes within the AVPV, little is known about the molecular mechanisms regulating their development. Previous work from our laboratory has demonstrated that Notch signaling is required in progenitor cell maintenance and formation of kisspeptin neurons of the arcuate nucleus (ARC) while simultaneously restraining POMC neuron number. Based on these findings, we hypothesized that the Notch signaling pathway may act similarly in the AVPV by promoting development of kisspeptin neurons at the expense of other neuronal subtypes. To address this hypothesis, we utilized a genetic mouse model with a conditional loss of Rbpj in Nkx2.1 expressing cells (Rbpj cKO). We noted an increase in cellular proliferation, as marked by Ki-67, in the hypothalamic ventricular zone (HVZ) in Rbpj cKO mice at E13.5. This corresponded to an increase in general neurogenesis and more TH-positive neurons. Additionally, an increase in OLIG2-positive early oligodendrocytic precursor cells was observed at postnatal day 0 in Rbpj cKO mice. By 5 weeks of age in Rbpj cKO mice, TH-positive cells were readily detected in the AVPV but few kisspeptin neurons were present. To elucidate the direct effects of Notch signaling on neuron and glia differentiation, an in vitro primary hypothalamic neurosphere assay was employed. We demonstrated that treatment with the chemical Notch inhibitor DAPT increased mKi67 and Olig2 mRNA expression while decreasing astroglial Gfap expression, suggesting Notch signaling regulates both proliferation and early glial fate decisions. A modest increase in expression of TH in both the cell soma and neurite extensions was observed after extended culture, suggesting that inhibition of Notch signaling alone is enough to bias progenitors towards a dopaminergic fate. Together, these data suggest that Notch signaling restricts early cellular proliferation and differentiation of neurons and oligodendrocytes both in vivo and in vitro and acts as a fate selector of kisspeptin neurons.

Complex integration of intrinsic and peripheral signaling is required for pituitary gland development.

The coordination of pituitary development is complicated and requires input from multiple cellular processes. Recent research has provided insight into key molecular determinants that govern cell fate specification in the pituitary. Moreover, increasing research aimed to identify, characterize, and functionally describe the presumptive pituitary stem cell population has allowed for a better understanding of the processes that govern endocrine cell differentiation in the developing pituitary. The culmination of this research has led to the ability of investigators to recapitulate some of embryonic pituitary development in vitro, the first steps to developing novel regenerative therapies for pituitary diseases. In this current review, we cover the major players in pituitary stem/progenitor cell function and maintenance, and the key molecular determinants of endocrine cell specification. In addition, we discuss the contribution of peripheral hormonal regulation of pituitary gland development, an understudied area of research.

Age- and sex-dependent effects of methamphetamine on cognitive flexibility and 5-HT2C receptor localization in the orbitofrontal cortex of Sprague-Dawley rats.

Adolescents and females experience worse outcomes of drug use compared to adults and males. This could result from age- and sex-specific consequences of drug exposure on brain function and cognitive behavior. In the current study, we examined whether a history of intravenous methamphetamine (METH) self-administration impacted cognitive flexibility and 5-HT2CR localization in the orbitofrontal cortex (OFC) in an age- and sex-dependent manner. Strategy shifting was assessed in male and female Sprague-Dawley rats that had self-administered METH (0.08 mg/kg/inf) or received non-contingent infusions of saline during periadolescence or young adulthood. After all rats reached adulthood, they were tested in an operant strategy shifting task and their brains were subsequently analyzed using immunofluorescence to quantify co-localization of 5-HT2C receptors with parvalbumin interneurons in the OFC. We found that adolescent-onset females were the only group impaired during discrimination and reversal learning, but they did not exhibit changes in localization of 5-HT2C receptors. In contrast, adult-onset males exhibited a significant increase in co-localization of 5-HT2C receptors within parvalbumin interneurons in the left hemisphere of the OFC. These studies reveal that age and sex differences in drug-induced deficits in reversal learning and 5-HT2CR co-localization with parvalbumin interneurons are dissociable and can manifest independently. In addition, these data highlight the potential for certain treatment approaches to be more suitable in some populations compared to others, such as alleviating drug-induced cognitive deficits as a focus for treatment in adolescent females.

Anterior Pituitary Transcriptome Suggests Differences in ACTH Release in Tame and Aggressive Foxes.

Domesticated species exhibit a suite of behavioral, endocrinological, and morphological changes referred to as "domestication syndrome." These changes may include a reduction in reactivity of the hypothalamic-pituitary-adrenal (HPA) axis and specifically reduced adrenocorticotropic hormone release from the anterior pituitary. To investigate the biological mechanisms targeted during domestication, we investigated gene expression in the pituitaries of experimentally domesticated foxes (Vulpes vulpes). RNA was sequenced from the anterior pituitary of six foxes selectively bred for tameness ("tame foxes") and six foxes selectively bred for aggression ("aggressive foxes"). Expression, splicing, and network differences identified between the two lines indicated the importance of genes related to regulation of exocytosis, specifically mediated by cAMP, organization of pseudopodia, and cell motility. These findings provide new insights into biological mechanisms that may have been targeted when these lines of foxes were selected for behavior and suggest new directions for research into HPA axis regulation and the biological underpinnings of domestication.

Effects of Exposure to the Endocrine-Disrupting Chemical Bisphenol A During Critical Windows of Murine Pituitary Development.

Critical windows of development are often more sensitive to endocrine disruption. The murine pituitary gland has two critical windows of development: embryonic gland establishment and neonatal hormone cell expansion. During embryonic development, one environmentally ubiquitous endocrine-disrupting chemical, bisphenol A (BPA), has been shown to alter pituitary development by increasing proliferation and gonadotrope number in females but not males. However, the effects of exposure during the neonatal period have not been examined. Therefore, we dosed pups from postnatal day (PND)0 to PND7 with 0.05, 0.5, and 50 µg/kg/d BPA, environmentally relevant doses, or 50 µg/kg/d estradiol (E2). Mice were collected after dosing at PND7 and at 5 weeks. Dosing mice neonatally with BPA caused sex-specific gene expression changes distinct from those observed with embryonic exposure. At PND7, pituitary Pit1 messenger RNA (mRNA) expression was decreased with BPA 0.05 and 0.5 µg/kg/d in males only. Expression of Pomc mRNA was decreased at 0.5 µg/kg/d BPA in males and at 0.5 and 50 µg/kg/d BPA in females. Similarly, E2 decreased Pomc mRNA in both males and females. However, no noticeable corresponding changes were found in protein expression. Both E2 and BPA suppressed Pomc mRNA in pituitary organ cultures; this repression appeared to be mediated by estrogen receptor-α and estrogen receptor-ß in females and G protein-coupled estrogen receptor in males, as determined by estrogen receptor subtype-selective agonists. These data demonstrated that BPA exposure during neonatal pituitary development has unique sex-specific effects on gene expression and that Pomc repression in males and females can occur through different mechanisms.

Isoliquiritigenin exhibits anti-proliferative properties in the pituitary independent of estrogen receptor function.

The plant flavonoid isoliquiritigenin (ISL) is a botanical estrogen widely taken as an herbal supplement to ease the symptoms of menopause. ISL has been also shown to have anti-tumor properties in a number of cancer cell backgrounds. However, the effects of ISL on normal cells are less well known and virtually unstudied in the context of the pituitary gland. We have established a pituitary explant culture model to screen chemical agents for gene expression changes within the pituitary gland during a period of active proliferation and differentiation. Using this whole-organ culture system we found ISL to be weakly estrogenic based on its ability to induce Cckar mRNA expression, an estrogen receptor (ER) mediated gene. Using a range of ISL from 200nM to 200µM, we discovered that ISL promoted cell proliferation at a low concentration, yet potently inhibited proliferation at the highest concentration. ICI 182,780 failed to antagonize ISL's repression of pituitary cell proliferation, indicating the effect is independent of ER signaling. Coincident with a decrease in proliferating cells, we observed down-regulation of transcript for cyclin D2 and E2 and a strong induction of mRNA and protein for the cyclin dependent kinase inhibitor Cdkn1a (p21). Importantly, high dose ISL did not alter the balance of progenitor vs. differentiated cell types within the pituitary explants and they seemed otherwise healthy; however, TUNEL staining revealed an increase in apoptotic cell death in ISL treated cultures. Our results merit further examination of ISL as an anti-tumor agent in the pituitary gland.

Identification of a novel progenitor cell marker, grainyhead-like 2 in the developing pituitary.

BACKGROUND: Pituitary stem/progenitor cells give rise to all of the endocrine cell types within the pituitary gland and are necessary for both development and gland homeostasis. Recent studies have identified several key factors that characterize the progenitor cell population. However, little is known about the factors that regulate progenitor cell differentiation and maintenance. Therefore, it is crucial to identify novel factors that help elucidate mechanisms of progenitor cell function in the developing pituitary. Our studies are the first to characterize the expression of Grainyhead-like 2 (GRHL2), a transcription factor known to regulate progenitor cell plasticity, in the developing pituitary. RESULTS: Our studies show GRHL2 expression is highest in the embryonic and early postnatal pituitary and is localized in pituitary progenitor cells. We demonstrate GRHL2 expression is changed in Notch2 cKO and Prop1df/df mice, mouse models that display progenitor cell number defects. In addition, our studies indicate a potential relationship between Notch signaling and GRHL2 expression in the developing pituitary. CONCLUSIONS: Taken together, our results indicate GRHL2 as a novel progenitor cell maker in the developing pituitary that may contribute to progenitor cell function and maintenance. Developmental Dynamics 245:1097-1106, 2016. © 2016 Wiley Periodicals, Inc.

Icam5 Expression Exhibits Sex Differences in the Neonatal Pituitary and Is Regulated by Estradiol and Bisphenol A.

Endocrine-disrupting chemicals are prevalent in the environment and can impair reproductive success by affecting the hypothalamic-pituitary-gonadal axis. The developing pituitary gland is sensitive to exposure to endocrine-disrupting chemicals, such as bisphenol A (BPA), and sex-specific effects can occur. However, effects on the critical window of neonatal pituitary gland development in mice have not been explored. Therefore, this study determined baseline gene expression in male and female pituitaries and consequences of environmental exposure to 17ß-estradiol (E2) and BPA on transcription of genes exhibiting sex differences during the neonatal period. Through microarray and quantitative RT-PCR analysis of pituitaries at postnatal day (PND)1, 3 genes were differentially expressed between males and females: Lhb, Fshb, and intracellular adhesion molecule-5 (Icam5). To see whether E2 and BPA exposure regulates these genes, pituitaries were cultured at PND1 with 10(-8) M E2 or 4.4 × 10(-6) M BPA. E2 decreased expression of Lhb, Fshb, and Icam5 mRNA in females but only significantly decreased expression of Icam5 in males. BPA decreased expression of Icam5 similarly to E2, but it did not affect Lhb or Fshb. Importantly, in vivo exposure to 50-µg/kg · d E2 from PND0 to PND7 decreased expression of Lhb, Fshb, and Icam5 mRNA in both males and females, whereas 50-mg/kg · d BPA exposure during the same time frame decreased expression of Icam5 in females only. Overall, we have uncovered that genes differentially expressed between the sexes can be regulated in part by hormonal and chemical signals in vivo and directly at the pituitary and can be regulated in a sex-specific manner.

Rbpj-κ mediated Notch signaling plays a critical role in development of hypothalamic Kisspeptin neurons.

The mammalian arcuate nucleus (ARC) houses neurons critical for energy homeostasis and sexual maturation. Proopiomelanocortin (POMC) and Neuropeptide Y (NPY) neurons function to balance energy intake and Kisspeptin neurons are critical for the onset of puberty and reproductive function. While the physiological roles of these neurons have been well established, their development remains unclear. We have previously shown that Notch signaling plays an important role in cell fate within the ARC of mice. Active Notch signaling prevented neural progenitors from differentiating into feeding circuit neurons, whereas conditional loss of Notch signaling lead to a premature differentiation of these neurons. Presently, we hypothesized that Kisspeptin neurons would similarly be affected by Notch manipulation. To address this, we utilized mice with a conditional deletion of the Notch signaling co-factor Rbpj-κ (Rbpj cKO), or mice persistently expressing the Notch1 intracellular domain (NICD tg) within Nkx2.1 expressing cells of the developing hypothalamus. Interestingly, we found that in both models, a lack of Kisspeptin neurons are observed. This suggests that Notch signaling must be properly titrated for formation of Kisspeptin neurons. These results led us to hypothesize that Kisspeptin neurons of the ARC may arise from a different lineage of intermediate progenitors than NPY neurons and that Notch was responsible for the fate choice between these neurons. To determine if Kisspeptin neurons of the ARC differentiate similarly through a Pomc intermediate, we utilized a genetic model expressing the tdTomato fluorescent protein in all cells that have ever expressed Pomc. We observed some Kisspeptin expressing neurons labeled with the Pomc reporter similar to NPY neurons, suggesting that these distinct neurons can arise from a common progenitor. Finally, we hypothesized that temporal differences leading to premature depletion of progenitors in cKO mice lead to our observed phenotype. Using a BrdU birthdating paradigm, we determined the percentage of NPY and Kisspeptin neurons born on embryonic days 11.5, 12.5, and 13.5. We found no difference in the timing of differentiation of either neuronal subtype, with a majority occurring at e11.5. Taken together, our findings suggest that active Notch signaling is an important molecular switch involved in instructing subpopulations of progenitor cells to differentiate into Kisspeptin neurons.