Understanding the physiology and pathology of an organ composed of a variety of cell populations depends critically on genome-wide information on each cell type. Here, we report single-cell transcriptome profiling of over 6,800 freshly dispersed anterior pituitary cells from postpubertal male and female rats. Six pituitary-specific cell types were identified based on known marker genes and characterized: folliculostellate cells and hormone-producing corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. Also identified were endothelial and blood cells from the pituitary capillary network. The expression of numerous developmental and neuroendocrine marker genes in both folliculostellate and hormone-producing cells supports that they have a common origin. For several genes, the validity of transcriptome analysis was confirmed by qRT-PCR and single cell immunocytochemistry. Folliculostellate cells exhibit impressive transcriptome diversity, indicating their major roles in production of endogenous ligands and detoxification enzymes, and organization of extracellular matrix. Transcriptome profiles of hormone-producing cells also indicate contributions toward those functions, while also clearly demonstrating their endocrine function. This survey highlights many novel genetic markers contributing to pituitary cell type identity, sexual dimorphism, and function, and points to relationships between hormone-producing and folliculostellate cells.

Continuous, as opposed to pulsatile, delivery of hypothalamic gonadotropin-releasing hormone (GnRH) leads to a marked decrease in secretion of pituitary gonadotropins LH and FSH and impairment of reproductive function. Here we studied the expression profile of gonadotropin subunit and GnRH receptor genes in rat pituitary in vitro and in vivo to clarify their expression profiles in the absence and continuous presence of GnRH. Culturing of pituitary cells in GnRH-free conditions downregulated Fshb, Cga, and Gnrhr expression, whereas continuous treatment with GnRH agonists upregulated Cga expression progressively and Gnrhr and Fshb expression transiently, accompanied by a prolonged blockade of Fshb but not Gnrhr expression. In contrast, Lhb expression was relatively insensitive to loss of endogenous GnRH and continuous treatment with GnRH, probably reflecting the status of Egr1 and Nr5a1 expression. Similar patterns of responses were observed in vivo after administration of a GnRH agonist. However, continuous treatment with GnRH stimulated LH secretion in vitro and in vivo, leading to decrease in LH cell content despite high basal Lhb expression. These data suggest that blockade of Fshb expression and depletion of the LH secretory pool are two major factors accounting for weakening of the gonadotroph secretory function during continuous GnRH treatment.

The mammalian pituitary gland is a complex organ consisting of hormone-producing cells (HPC), nonhormonal folliculostellate cells (FSC) and pituicytes, vascular pericytes and endothelial cells, and putative Sox2-expressing stem cells. Here, we used scRNAseq analysis of adult female rat pituitary cells to study the heterogeneity of pituitary cells with a focus on evaluating the transcriptomic profile of the Sox2expressing population. Samples containing whole pituitary and separated anterior and posterior lobe cells allowed the identification of all expected pituitary resident cell types and lobe-specific subpopulations of vascular cells. Sox2 was expressed uniformly in all FSC, pituicytes, and a fraction of HPC. FSC comprised two subclusters; FSC1 contained more cells but expressed less genetic diversity compared to FSC2. The latter contained proliferative cells, expressed genes consistent with stem cell niche formation, including tight junctions, and shared genes with HPC. The FSC2 transcriptome profile was also consistent with the activity of pathways regulating cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. The expression of other stem cell marker genes was common for FSC and pituicytes (Sox9,

Triiodothyronine (T3) is an important modulator of cardiac metabolism and function, often through modulation of gene expression. The cardiomyocyte circadian clock is a transcriptionally-based molecular mechanism capable of regulating cardiac processes, in part by modulating responsiveness of the heart to extra-cardiac stimuli/stresses in a time-of-day- (TOD) dependent manner. Although TOD-dependent oscillations in circulating levels of T3 (and its intermediates) have been established, whether oscillations in T3 sensitivity in the heart occur is unknown. To investigate the latter possibility, euthyroid male Wistar rats were treated with vehicle or T3 at distinct times of the day, after which induction of known T3 target genes were assessed in the heart (4-h later). The expression of mRNA was assessed by Real-Time qPCR. Here, we report greater T3 induction of transcript levels at the end of the dark phase. Surprisingly, use of cardiomyocyte-specific clock mutant (CCM) mice revealed that TOD-dependent oscillations in T3 sensitivity were independent of this cell autonomous mechanism. Investigation of genes encoding for proteins that affect T3 sensitivity revealed that Dio1, Dio2, and Thrb1 exhibited TOD-dependent variations in the heart, while Thra1 and Thra2 did not. Of these, Dio1 and Thrb1 were increased in the heart at the end of the dark phase. Interestingly, we observed that T3 acutely altered the expression of core clock components (e.g., Bmal1) in the rat heart. To investigate this further, rats were injected with a single dose of T3, after which expression of clock genes were interrogated at 3-h intervals over the subsequent 24h-period. These studies revealed robust effects of T3 on oscillations of both core clock components and clock-controlled genes. In summary, the current study exposed time-of-day-dependent rhythms in cardiac T3 sensitivity, and that T3 alters the circadian clock in the heart.

The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs – which phosphorylate PI(4, 5)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.

Cell-matrix interactions play important roles in pituitary development, physiology, and pathogenesis. In other tissues, a family of non-collagenous proteins, termed SIBLINGs, are known to contribute to cell-matrix interactions. Anterior pituitary gland expresses two SIBLING genes, Dmp1 (dentin matrix protein-1) and Spp1 (secreted phosphoprotein-1) encoding DMP1 and osteopontin proteins, respectively, but their expression pattern and roles in pituitary functions have not been clarified. Here we provide novel evidence supporting the conclusion that Spp1/ osteopontin, like Dmp1 /DMP1, are expressed in gonadotrophs in a sex- and age-specific manner. Other anterior pituitary cell types do not express these genes. In contrast to Dmp1, Spp1 expression is higher in males; in females, the expression reaches the peak during the diestrus phase of estrous cycle. In further contrast to Dmp1 and marker genes for gonadotrophs, the expression of Spp1 is not regulated by gonadotropin-releasing hormone in vivo and in vitro . However, Spp1 expression increases progressively after pituitary cell dispersion in both female and male cultures. We may speculate that gonadotrophs signal to other pituitary cell types about changes in the structure of pituitary cell-matrix network by osteopontin, a function consistent with the role of this secretory protein in postnatal tissue remodeling, extracellular matrix reorganization after injury, and tumorigenesis.

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