Gwen V. Childs

Introduction: Estrogen’s broad trophic effects on anterior pituitary function are not fully understood. The compound 4-vinyl cyclohexene diepoxide (VCD) induces early ovarian failure and reduced E2 production, which may differentially affect anterior pituitary cells. We recently identified transcriptomic changes in pituitary gonadotropes at the single-cell level in females treated with VCD for 20 days, which were acyclic, with low serum estradiol (E2) levels and 8-12-fold increases in gonadotropins 138 days later. Materials and methods: We tested vehicle-control and VCD-treated female mice in metabolic cages and assayed serum hormone levels by EIA, and pituitary cell transcripts by qRT-PCR and single-cell RNA-seq. Results: VCD-treated mice showed normal metabolic health. Compared with controls, VCD-treated females exhibited a 5-fold elevation in serum pituitary thyroid-stimulating hormone (TSH) levels and near-normal thyroid hormone levels. Pituitary Tshb mRNA levels were reduced, and pituitary Gh, Prl , and Pomc mRNA levels were unchanged. Serum growth hormone (GH) levels were higher in VCD-treated mice than in controls 62 days after the start of treatment (perimenopause) and then reduced to control levels after 118–138 days. Ingenuity Pathway Analyses (IPA) of upregulated differentially expressed gene (DEG) transcripts predicted activation of G protein-mediated signaling pathways in one scRNA-seq somatotrope subcluster and in the thyrotrope and lactotrope clusters. In contrast, downregulated DEGs in corticotropes and the remaining two somatotrope clusters predicted downregulation of critical signaling pathways. Discussion: The unchanged pituitary Gh, Prl , and Pomc mRNA levels correlated with unchanged serum levels. The interim rise in serum GH 62 days after treatment agrees with reports of perimenopausal responses due to estrogen spikes. Reduced pituitary Tshb correlates with the recently reported reduced expression in gonadotropes. IPA analysis of upregulated DEGs in thyrotrope clusters predicted the activation of canonical signaling pathways that support elevated serum TSH levels. The near-normal thyroid hormone levels suggest subclinical hypothyroidism in menopausal women. Downregulated DEGs in corticotropes and in two somatotrope subclusters predicted compromised stress and metabolic responses. Finally, reduced multihormone gene expression suggested compromised plasticity in a low-estrogen environment. These different responses to a sustained low-estrogen state, such as that seen in menopause, reflect the diversity of estrogen’s trophic actions.

The RNA-binding proteins Musashi1 and Musashi2 (MSI1 and MSI2) regulate stem cell function and tissue plasticity by modulating mRNA translation. While typically known as translational repressors, the MSI1 and MSI2 proteins can also act as context-dependent activators of mRNA translation, although the mechanism of MSI-mediated translational activation is unknown. Here, we identify Embryonic Lethal Abnormal Vision-like (ELAVL) proteins as essential co-regulators of MSI1-dependent translational activation. In Xenopus laevis oocytes, antisense oligonucleotide knockdown of Elavl4 inhibited progesterone-stimulated maturation and blocked polyadenylation and translation of key MSI target mRNAs, including the Mos and Cyclin B5 mRNAs. Exogenous expression of ELAVL4 rescued these defects, confirming its necessity for maturation and cell cycle progression. Mechanistically, we determined that the ELAVL4 C-terminal domain interacts with the N-terminal RNA recognition motifs of MSI1 in an RNA-independent manner. Mass spectrometry and functional assays revealed this interaction is evolutionarily conserved: mouse ELAVL1 interacts with MSI1 in the pituitary, and human ELAVL1 rescues Elavl4-depleted Xenopus oocytes. Furthermore, knockdown of Elavl1 in a mammalian cell line abrogated MSI-dependent translational activation of a pituitary Prop1 3′ UTR mRNA reporter. Our results establish a conserved mechanism where ELAVL family members interact with MSI to promote MSI-dependent mRNA translational activation.

The RNA-binding proteins Musashi1 and Musashi2 (MSI1 and MSI2) regulate stem cell function and tissue plasticity by modulating mRNA translation. While typically known as translational repressors, the MSI1 and MSI2 proteins can also act as context-dependent activators of mRNA translation, although the mechanism of MSI-mediated translational activation are unknown. Here, we identify Embryonic Lethal Abnormal Vision-like (ELAVL) proteins as essential co-regulators of MSI1-dependent translational activation. In Xenopus laevis oocytes, antisense oligonucleotide knockdown of Elavl4 inhibited progesterone-stimulated maturation and blocked polyadenylation and translation of key MSI target mRNAs, including the Mos and Cyclin B5 mRNAs. Exogenous expression of ELAVL4 rescued these defects, confirming its necessity for maturation and cell cycle progression. Mechanistically, we determined that the ELAVL4 C-terminal domain interacts with the N-terminal RNA recognition motifs of MSI1 in an RNA-independent manner. Mass spectrometry and functional assays revealed this interaction is evolutionarily conserved: mouse ELAVL1 interacts with MSI1 in the pituitary, and human ELAVL1 rescues Elavl4-depleted Xenopus oocytes. Furthermore, knockdown of Elavl1 in a mammalian cell line abrogated MSI-dependent translational activation of a pituitary Prop1 3’ UTR mRNA reporter. Our results establish a conserved mechanism where ELAVL family members interact with MSI to promote MSI-dependent mRNA translational activation.

To study the effect of low-estrogen and the menopause state upon female pituitary function, we used the 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure model to gradually reduce serum estradiol (E2) levels. We utilized single-cell RNA sequencing transcriptomics analysis (scRNA-seq) to determine how reduced E2 levels influence pituitary gonadotrope gene expression and cell state, with a focus on potential contributions from pituitary stem cells to gonadotrope population growth. VCD-treated mice were acyclic and exhibited 8–15-fold increases in serum levels of the gonadotropins follicle-stimulating hormone and luteinizing hormone. Serum E2 levels were half those of normally cycling proestrous females. Pituitary scRNA-seq identified two gonadotrope populations based upon distinct gene expression signatures (a higher cell number primary cluster, designated here as GON1 and a smaller cell number secondary cluster, designated GON2). The GON1 population increased in number significantly in low-E2, VCD-treated mice, expressing markers indicative of contributions from pituitary stem cell activation. Both gonadotrope populations showed upregulated genes for canonical pathways supporting mRNA translation and hormone secretion, which correlated well with the high serum gonadotropin levels. Consistent with the high serum gonadotropin levels, Fshb and Lhb mRNAs were increased in whole pituitary samples (quantitative real time polymerase chain reaction). The pituitary Sox2-positive stem cell population exhibited increased expression of more mature transitional progenitors, including Sox9 mRNA, and showed upregulation of pathways involved in stem cell development and mitotic activity. This model provides insight into the cellular and molecular adaptations of the pituitary to the low-E2, menopausal state, with potential relevance for understanding human perimenopausal and menopausal physiology.

Introduction: Somatotropes lacking leptin receptors (LEPR) produce less growth hormone and are poorly responsive to growth hormone releasing hormone (GHRH). Transcriptomic analysis reveals that the mutant somatotropes contain progenitor cell markers (Sox9+) and multiple pituitary hormone transcripts-(Pomc, Prl, Lhb, Tshb and Cga), suggesting that the cells are progenitor cells. The resulting GH deficiency contributes to adult-onset obesity in the mutant, due to an increase in abdominal fat.Objectives: This study examined how a high-fat diet (HFD) affected pituitary transcriptomic function in older (10-month) female mutants lacking leptin receptors (LEPR) in somatotropes and intact littermate controls. We hypothesized that pituitary cells from both the older control females and the female mutants would be greatly affected by the oxidative stress from the HFD.Methods: Mice were exposed to a 60% HFD for 16 weeks, followed by glucose tolerance testing and 3-day monitoring in metabolic cages (CLAMS). Pituitaries were harvested, cells dispersed and subjected to single cell-RNA-seq (scRNA-seq) with bioinformatic analysis. Serum was collected and analyzed for pituitary hormones and cytokines.Results: The HFD resulted in elevated serum leptin and IL-6 in both mutants and controls, and reduced serum growth hormone (GH) and prolactin (PRL) levels. However, adrenocorticotropin (ACTH) levels were elevated in controls but not mutants. Unexpectedly, whereas controls gained as much weight as younger females, somatotrope LEPR-null mutants on a HFD gained only 75% of the weight of controls, were more glucose tolerant, consumed less food, were more active in the metabolic cages, and had lower serum levels of insulin. Analysis of scRNA-seq revealed that the HFD induced differentially expressed genes (DEGs) in more distinct pituitary cell populations of older mice compared to previously reported findings in younger control females, indicating greater vulnerability in the older pituitary population. This was especially true in the mutant pituitary population. Ingenuity Pathway Analysis indicated that the DEGs included targets of critical upstream regulators important for pituitary cell function and plasticity (CREB, Fox01, cAMP, STAT3, insulin, TRH, GnRH, and leptin signaling pathways), with most pathways predicted to be downregulated by the HFD. Unlike controls, HFD-fed mutant cell populations exhibited DEGs consistent with the downregulation of translational regulatory pathways. Notably, the HFD reversed the increased expression of progenitor cell markers (Sox9+) and multiple pituitary hormone transcripts seen in the mutant on a control diet. Similarly, the HFD also reversed the expression of multiple pituitary hormone transcripts and progenitor markers in lactotropes, thyrotropes, and corticotropes from mutants.Conclusion: The findings supported our hypothesis that both aging and the mutation (loss of LEPR in somatotropes) would render these mice more sensitive to a HFD as more pituitary cell types were affected transcriptionally. Collectively, these findings indicate that HFD and/or obese state may compromise pituitary plasticity by down-regulating translational processes and reducing expression in cells that may have multipotential functions. The oxidative stress of a HFD may thus limit the expression of pituitary progenitor cells.

Anterior pituitary somatotropes respond to metabolic signals from the adipokine leptin to optimize functional responses to the body's nutritional state via growth hormone (GH) secretion. Molecular targets of leptin in pituitary somatotropes include GH, the GH-releasing hormone receptor (GHRHR), and, in females, the transcription factor POU1F1, all of which are dependent on leptin stimulation for expression. To identify the trophic mechanisms underlying leptin action upon somatotropes, we analyzed single-cell gene transcriptomes comparing pituitaries from a female mouse model bearing somatotropes lacking leptin receptors (LEPR-null mutants) and control pituitaries. Computational clustering of results identified all common pituitary cell types and differentially expressed genes. Mutant female somatotrope clusters showed decreased levels of Gh and Htatsf1 mRNA, which was also reduced in mutant pituitaries lacking Prop1 or POU1F1. Mutant somatotropes also showed increased expression of markers for pituitary stem and progenitor cells (eg, Sox9) and increased (1.73-6.7 fold) expression of nonsomatotrope hormones, Pomc, Lhb, Tshb, Cga, and Prl. Conversely, the mutant female Sox2-positive stem cell cluster showed decreased expression of markers for stem cells and increased expression of pituitary hormone genes. The data support a model in which the female pituitary somatotrope cell population's development and/or maintenance requires leptin trophic signals and also suggests that, in the absence of normal somatotrope maturation, pituitary stem cells are driven towards premature differentiation.

The pituitary functions as a master endocrine gland that secretes hormones critical for regulation of a wide variety of physiological processes including reproduction, growth, metabolism and stress responses. The distinct hormone-producing cell lineages within the pituitary display remarkable levels of cell plasticity that allow remodeling of the relative proportions of each hormone-producing cell population to meet organismal demands. The molecular mechanisms governing pituitary cell plasticity have not been fully elucidated. Our recent studies have implicated a role for the Musashi family of sequence-specific mRNA binding proteins in the control of pituitary hormone production, pituitary responses to hypothalamic stimulation and modulation of pituitary transcription factor expression in response to leptin signaling. To date, these actions of Musashi in the pituitary appear to be mediated through translational repression of the target mRNAs. Here, we report Musashi1 directs the translational activation, rather than repression, of the Prop1, Gata2 and Nr5a1 mRNAs which encode key pituitary lineage specification factors. We observe that Musashi1 further directs the translational activation of the mRNA encoding the glycolipid Neuronatin (Nnat) as determined both in mRNA reporter assays as well as in vivo. Our findings suggest a complex bifunctional role for Musashi1 in the control of pituitary cell function.

Anterior pituitary cell function requires a high level of protein synthesis and secretion which depend heavily on mitochondrial adenosine triphosphate production and functional endoplasmic reticula. Obesity adds stress to tissues, requiring them to adapt to inflammation and oxidative stress, and adding to their allostatic load. We hypothesized that pituitary function is vulnerable to the stress of obesity. Here, we utilized a 10- to 15-week high-fat diet (HFD, 60%) in a thermoneutral environment to promote obesity, testing both male and female FVB.129P mice. We quantified serum hormones and cytokines, characterized the metabolic phenotype, and defined changes in the pituitary transcriptome using single-cell RNA-sequencing analysis. Weight gain was significant by 3 weeks in HFD mice, and by 10 weeks all HFD groups had gained 20 g. HFD females (15 weeks) had increased energy expenditure and decreased activity. All HFD groups showed increases in serum leptin and decreases in adiponectin. HFD caused increased inflammatory markers: interleukin-6, resistin, monocyte chemoattractant protein-1, and tumor necrosis factorα. HFD males and females also had increased insulin and increased TSH, and HFD females had decreased serum prolactin and growth hormone pulse amplitude. Pituitary single-cell transcriptomics revealed modest or no changes in pituitary cell gene expression from HFD males after 10 or 15 weeks or from HFD females after 10 weeks. However, HFD females (15 weeks) showed significant numbers of differentially expressed genes in lactotropes and pituitary stem cells. Collectively, these studies reveal that pituitary cells from males appear to be more resilient to the oxidative stress of obesity than females and identify the most vulnerable pituitary cell populations in females.

Disclosure: A. Herdman: None. A. Lagasse: None. A. Haney: None. S. Byrum: None. U. Boehm: None. M.C. MacNicol: None. A.M. MacNicol: None. G.V. Childs: None. A.K. Odle: None. Secretoneurin (SN) is a proteolytic product of secretogranin II (SCG2), a member of the granin family of proteins. Both SCG2 and SN have been localized in the anterior pituitary (AP), and SCG2 co-localizes to gonadotrope granules containing luteinizing hormone (LH). Preliminary studies from others have shown that exogenous SN is capable of stimulating LH secretion from the mouse LßT2 cell line and goldfish pituitaries, and SN may provide a paracrine signal from other AP cells to support LH production/secretion. Despite the implications for regulatory reproductive signaling, a comprehensive study of the effect of SN on female mammalian gonadotropes has not been conducted. To define the pathways and processes affected by exogenous secretoneurin (SN) on murine gonadotrope protein expression, pituitaries from 18 proestrous female mice expressing Gnrhr-driven Cre and a floxed fluorescent Cre reporter were collected and dispersed. Half of the dispersed pituitaries were stimulated with 1 μM SN, and all cells were incubated for 3 hours. Gonadotropes were purified via fluorescence-activated cell sorting. After pooling to optimize cell counts, control and SN-stimulated gonadotropes were submitted for mass-spectrometry proteomics. A differential expression analysis was performed to determine fold-change (FC) in proteins from SN-stimulated samples over control (unstimulated), and false discovery rate (FDR)-controlled adjusted p values were used to assign significance (Adj p≤0.05). A total of 948 proteins were identified in the purified gonadotrope pools. Of these, 136 proteins were significantly differentially expressed between control and SN-stimulated gonadotrope pools; 53 proteins were upregulated and 83 were downregulated in SN-stimulated gonadotropes. Upregulated targets of particular interest include the SN precursor SCG2 (FC = 12, pCga mRNA following SN stimulation. In addition to the expected “cell signaling” and “response to stimulation”, gene ontology analysis showed that the major biological processes altered by SN stimulation include processing of RNA (including RNA binding, translation regulation, etc.) and regulation of cell structure. Indeed, several RNA-binding proteins and actin-related proteins were significantly changed. Our data confirm previous results from LßT2 cells and define new SN targets and pathways that may play a broader role in gonadotrope physiology. Presentation: Friday, June 16, 2023