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Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

ABSTRACT: Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-h?CGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches.

SUBMITTER: Murtazina DA

PROVIDER: S-EPMC7443347 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

Murtazina Dilyara A DA Arreguin-Arevalo Jesus Alejandro JA Cantlon Jeremy D JD Ebrahimpour-Boroojeny Ali A Shrestha Akash A Hicks Jennifer A JA Magee Christianne C Kirkley Kelly K Jones Kenneth K Nett Terry M TM Chitsaz Hamidreza H Clay Colin M CM

Biology of reproduction 20200201 1

Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we ex ...[more]

PMID: 31504222

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Project description:The goals of this study is to enrich the population of ovine gonadotropes via adenovirus-mediated targeting and flow cytometry to compare NGS-derived transcriptome profiling (RNA-seq) of a gonadotrope-enriched cell population to the combined transcriptome of all anterior pituitary cells and apply enrichment method to query the transcriptome of sorted ovine pituitary cells in response to treatment with 10 nM estradiol (E2) for six hours. Methods: mRNA profiles of sheep anterior pituitary cells were generated by deep sequencing, in sextuplicates, using Illumina HiSeq. The sequence reads that passed quality filters were analyzed at the transcript level with GSNAP, followed by featureCounts and then followed by edgeR. qRT–PCR validation was performed using SYBR Green assays. Results: About 124 million sequence reads per sample were mapped to the sheep genome (Oar v.3.1) which identified 17,082 transcripts in the pituitaries. RNA-seq data confirmed overrepresentation of genes known to be expressed in pituitary in sorted samples; 11 of genes (5 gonadotrope-specific and 6 non gonadotrope-specific) were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR. Approximately 7.2% of the transcripts showed differential expression between the sorted (gonadotropes) and unsorted (all anterior pituitary cells) pituitary cells, with a fold change ≥3 and FDR <0.05. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to gonadotrope function. 232 genes were detected as upregulated in the sorted GFP-positive cells treated with E2 (gonadotropes) and 31 were downregulated following E2 treatment with a fold change ≥1.5 and FDR <0.1. Conclusions: Our study represents the first detailed analysis of the gonadotrope transcriptome, with 6 biologic replicates, generated by RNA-seq technology. The data analysis workflows reported here should provide a framework for comparative investigations of gonadotrope expression profiles. This method was applied to determine differentially expressed genes in the sorted population of pituitary cells (gonadotropes) in response to 10 nM E2.

Project description:The beta subunit of follicle stimulating hormone (FSHB) is expressed specifically in pituitary gonadotropes in vertebrates. Transgenic mouse studies have shown that enhancers in the proximal promoter between -172/-1 bp of the ovine FSHB gene are required for gonadotrope expression of ovine FSHB. These enhancers are associated with regulation by activins and gonadotropin releasing hormone (GnRH). Additional distal promoter sequence between -4741/-750 bp is also required for expression. New transgenic studies presented here focus on this distal region and narrow it to 1116 bp between -1866/-750 bp. In addition, adenoviral constructs were produced to identify these critical distal sequences using purified primary mouse gonadotropes as an in vitro model system. The adenoviral constructs contained -2871 bp, -750 bp or -232 bp of the ovine FSHB promoter. They all showed gonadotrope-specific regulation since they were induced only in purified primary gonadotropes by activin A (50 ng/ml) and inhibited by GnRH (100 nM) in the presence of activin (except -232FSHBLuc). However, basal expression of all three viral constructs (in the presence of follistatin to block cellular induction by activin) was relatively high in pituitary non-gonadotropes as well as gonadotropes. Thus, gonadotrope-specific regulation associated with the proximal promoter was observed as expected, but the model was blind to distal promoter elements between -2871/-750 necessary for gonadotrope-specific expression of ovine FSHB in vivo. The new adenoviral-based in vitro technique did detect, however, a novel GnRH response element between -750 bp and -232 bp of the ovine FSHB promoter. We conclude that adenoviral-based studies in primary gonadotropes can adequately recognize regulatory elements on the ovine FSHB promoter associated with gonadotrope-specific regulation/expression, but that more physiologically based techniques, such as transgenic studies, will be needed to identify sequences between -1866/-750 bp of the ovine FSHB promoter that are also required for tissue/cell specific expression in vivo.

Dataset Information

Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

Publications

Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

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