Single nucleus transcriptome and chromatin accessibility of postmortem human pituitaries reveal diverse stem cell regulatory mechanisms.

Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus RNA-seq and ATAC-seq resource from pediatric, adult, and aged postmortem pituitaries (snpituitaryatlas.princeton.

Single nucleus multi-omics regulatory landscape of the murine pituitary.

To provide a multi-omics resource and investigate transcriptional regulatory mechanisms, we profile the transcriptome, chromatin accessibility, and methylation status of over 70,000 single nuclei (sn) from adult mouse pituitaries. Paired snRNAseq and snATACseq datasets from individual animals highlight a continuum between developmental epigenetically-encoded cell types and transcriptionally-determined transient cell states. Co-accessibility analysis-based identification of a putative Fshb cis-regulatory domain that overlaps the fertility-linked rs11031006 human polymorphism, followed by experimental validation illustrate the use of this resource for hypothesis generation.

Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines.

LT2 and T3-1 are important, widely studied cell line models for the pituitary gonadotropes that were generated by targeted tumorigenesis in transgenic mice. LT2 cells are more mature gonadotrope precursors than T3-1 cells. Microsatellite authentication patterns, chromosomal characteristics, and their intercellular variation have not been reported.

Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity.

Immediate-early response genes (IEGs) are rapidly and transiently induced following an extracellular signal. Elucidating the IEG response patterns in single cells (SCs) requires assaying large numbers of timed samples at high accuracy while minimizing handling effects. To achieve this, we developed and validated RNA stabilization Buffer for Examination of Single-cell Transcriptomes (RNA-Best), a versatile single-step cell and tissue preservation protocol that stabilizes RNA in intact SCs without perturbing transcription patterns.

Regulatory Architecture of the LβT2 Gonadotrope Cell Underlying the Response to Gonadotropin-Releasing Hormone.

The LβT2 mouse pituitary cell line has many characteristics of a mature gonadotrope and is a widely used model system for studying the developmental processes and the response to gonadotropin-releasing hormone (GnRH). The global epigenetic landscape, which contributes to cell-specific gene regulatory mechanisms, and the single-cell transcriptome response variation of LβT2 cells have not been previously investigated. Here, we integrate the transcriptome and genome-wide chromatin accessibility state of LβT2 cells during GnRH stimulation.

Modeling and high-throughput experimental data uncover the mechanisms underlying gene sensitivity to gonadotropin-releasing hormone pulse frequency.

Neuroendocrine control of reproduction by brain-secreted pulses of gonadotropin-releasing hormone (GnRH) represents a longstanding puzzle about extracellular signal decoding mechanisms. GnRH regulates the pituitary gonadotropin's follicle-stimulating hormone (FSH) and luteinizing hormone (LH), both of which are heterodimers specified by unique β subunits (FSHβ/LHβ). Contrary to , gene induction has a preference for low-frequency GnRH pulses.

Growth differentiation factor 9 (GDF9) forms an incoherent feed-forward loop modulating follicle-stimulating hormone β-subunit (FSHβ) gene expression.

Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses from the hypothalamus and regulates follicle-stimulating hormone β-subunit (FSHβ) gene expression in pituitary gonadotropes in a frequency-sensitive manner. The mechanisms underlying its preferential and paradoxical induction of FSHβ by low frequency GnRH pulses are incompletely understood. Here, we identify growth differentiation factor 9 (GDF9) as a GnRH-suppressed autocrine inducer of FSHβ gene expression.

Outside the box signaling: secreted factors modulate GnRH receptor-mediated gonadotropin regulation.

Control of gene expression following activation of membrane receptors results from the regulation of intracellular signaling pathways and transcription factors. Accordingly, research to elucidate the regulatory control circuits and cellular data processing mechanisms focuses on intracellular mechanisms. While autocrine and paracrine signaling are acknowledged in endocrinology, secreted factors are not typically recognized as fundamental components of the pathways connecting cell surface receptors to gene control in the nucleus.

Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes.

Identifying the early gene program induced by GnRH would help understand how GnRH-activated signaling pathways modulate gonadotrope secretory response. We previously analyzed GnRH-induced early genes in LβT2 cells, however these lack GnRH self-potentiation, a physiological attribute of gonadotropes. To minimize cellular heterogeneity, rat primary pituitary cultures were enriched for gonadotropes by 40-60% using a sedimentation gradient.

Ca2+-activated K+ channels in gonadotropin-releasing hormone-stimulated mouse gonadotrophs.

GnRH receptor activation elicits release of intracellular Ca(2+), which leads to secretion and also activates Ca(2+)-activated ion channels underlying membrane voltage changes. The predominant Ca(2+)-activated ion channels in rat and mouse gonadotrophs are Ca(2+)-activated K(+) channels. To establish the temporal relationship between GnRH-induced changes in intracellular [Ca(2+)] ([Ca(2+)](i)) and membrane current (I(m)), and to identify specific Ca(2+)-activated K(+) channels linking GnRH-induced increase in [Ca(2+)](i) to changes in plasma membrane electrical activity, we used single female mouse gonadotrophs in the perforated patch configuration of the patch-clamp technique, which preserves signaling pathways.

Differential expression and regulation of progesterone receptor isoforms in rat and mouse pituitary cells and LbetaT2 gonadotropes.

Manipulation of endogenous progesterone receptor (PR) does not produce equivalent physiological effects in mouse and rat pituitary cells. To test whether this may be due in part to difference in PR isoform expression, we examined hormonally regulated pituitary PR-A and PR-B mRNA levels using quantitative real-time PCR. The LbetaT2 mouse gonadotrope line or pituitary cells from adult, ovariectomized rats or mice were cultured with or without 0.

Estradiol inhibition of voltage-activated and gonadotropin-releasing hormone-induced currents in mouse gonadotrophs.

We report the first study of voltage-activated and GnRH-induced plasma membrane currents and their modulation by estradiol (E2) in mouse gonadotrophs. In consideration of the pleiotropic effects of E2 on gonadotrophin secretion and the relationship between plasma membrane electrical excitability and secretion, our objective was to determine the role of E2 in modulating gonadotroph plasma membrane currents. We measured total voltage-activated and GnRH-induced currents using the perforated-patch configuration of the patch-clamp technique, which preserves signaling pathways, including GnRH-induced Ca2+ oscillations.

Complex actions of sex steroids in adipose tissue, the cardiovascular system, and brain: Insights from basic science and clinical studies.

Recent publications describing the results of the Women's Health Initiative (WHI) and other studies reporting the impact of hormone therapy on aging women have spurred reexamination of the broad use of estrogens and progestins during the postmenopausal years. Here, we review the complex pharmacology of these hormones, the diverse and sometimes opposite effects that result from the use of different estrogenic and progestinic compounds, given via different delivery routes in different concentrations and treatment sequence, and to women of different ages and health status. We examine our new and growing appreciation of the role of estrogens in the immune system and the inflammatory response, and we pose the concept that estrogen's interface with this system may be at the core of some of the effects on multiple physiological systems, such as the adipose/metabolic system, the cardiovascular system, and the central nervous system.

Hormone therapy: physiological complexity belies therapeutic simplicity.

The results of the Women's Health Initiative, a study anticipated to provide definitive answers about health benefits and risks of postmenopausal hormone therapy, have generated debate and confusion among clinicians, researchers, and the lay public. The ovarian hormones estrogen and progesterone, which decline at menopause, normally elicit complex tissue-specific responses throughout the body. Major advances are providing a detailed molecular definition of how that differential action is achieved.