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Single cell RNA sequencing of the steroidogenic lineage in the male adrenal cortex

ABSTRACT: To achieve single-cell characterization of the adrenal cortex transcriptome, we selectively labeled the steroidogenic adrenocortical cells using a transgenic Cre-LoxP approach. We combined Sf1-Crehigh mice with Rosa26mTmG animals to obtain mice expressing fluorescent reporter proteins at the cell membrane (mTmG) of cells that express or have expressed the transcription factor SF-1 (Steroidogenic Factor 1, also AdBP4, encoded by NR5A1). We prepared a single-cell suspension by combining mechanic and enzymatic dissociation at low temperatures to preserve the viability of steroid cells and their transcriptional states. Fluorescence-activated cell sorting (FACS) was then used to isolate GFP-positive cells representing the steroidogenic lineage. GFP-positive cells include the progenitor populations as well as differentiated cells (zona glomerulosa and zona fasciculata cells).

ORGANISM(S): Mus musculus

PROVIDER: GSE291343 | GEO | 2025/12/01

REPOSITORIES: GEO

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Project description:The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female -induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.

Project description:In order to analyze the transcriptome characteristics of aldosterone producing cell clusters (APCC) we compared transcript abundances of APCC, zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors. The frozen adrenal glands in O.C.T. compound were cut into 7um sections, and every 10-th section immunostained for aldosterone synthase (CYP11B2). The remaining sections were stained with cresyl violet and used for laser-capture microdissection of tissue to use in the array assays. APCC and ZG samples were captured from CYP11B2 positive regions based on the CYP11B2-stained sections. ZF and ZR were captured from lipid-rich cells in the middle layer and compact cells outside of the medulla, respectively. RNA was isolated using PicoPure RNA isolation kits (Molecular Devices, Sunnyvale, CA). 1-10 ng total RNA was reverse-transcribed and amplified with the Ovation Pico WTA System V2 (NuGEN Technologies, San Carlos, CA). cDNA was purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and biotin-labeled using Encore Biotin Module (NuGEN Technologies), followed by hybridization to GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). Expression values were calculated using the robust multi-array average method (RMA). This resulted in base-2 log-transformed data for each of the 4 tissues from each of the 4 people. In addition to the raw and processed data we also supply a supplementary Excel file holding the data and some statistical analysis, which has features to make simple graphs, and holds probe-set annotation that we used at that time (users may wish to obtain new annotation though). We fit two-way ANOVA models with terms for 4 tissues and 4 people, and compared each probe-set between every pair of tissues using F-tests for pairwise contrasts. We modeled people effects since they were not negligible. The supplement shows how to calculate the tests. Aldosterone producing cell clusters (APCC), zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors, were individually assayed on 16 Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Expression values were estimated with the Robust Multi-Arry average (RMA) algorithm, which resulted in log-2 transformed values for each of 54675 probe sets.

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Single cell RNA sequencing of the steroidogenic lineage in the male adrenal cortex

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