HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

Vijay Simha Baddela,Arpna Sharma,Marten Michaelis,Jens Vanselow

doi:10.1038/s41598-020-60935-1

Vijay Simha Baddela, Arpna Sharma + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-60935-1

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Abstract

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor, consisting of a constitutively expressed β-subunit (HIF1B) and a regulated α-subunit (HIF1A). In the present study, we analyzed the HIF1 driven transcriptional activity in bovine granulosa cells (GC). Treatment of GC with FSH (follicle stimulating hormone) and IGF1 (insulin-like growth factor 1) resulted in the upregulation of HIF1A mRNA expression under normoxia. Immunohistochemistry of bovine ovarian sections showed distinct staining of HIF1A in the GC layer of different staged ovarian follicles. Suppression of HIF1 using echinomycin and gene knockdown procedures revealed that HIF1 transcriptionally regulates the genes associated with steroidogenesis (STAR, HSD3B and CYP19A1) and proliferation (CCND2 and PCNA) of GC. Further, our data suggest that CYP19A1, the key gene of estradiol production, is one of the plausible downstream targets of HIF1 in bovine GC as shown by gene expression, radioimmunoassay, and chromatin precipitation analysis. Based on these results, we propose that HIF1 driven transcriptional activity plays a crucial role in GC functionality, especially steroidogenesis and proliferation in developing bovine ovarian follicles.

Highlights

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor, consisting of a constitutively expressed β-subunit (HIF1B) and a regulated α-subunit (HIF1A)
It has been implied that Wnt induced phosphatidylinositol 3-kinase (PI3k)/Akt signaling and signal transducers and activators of transcription 3 (STAT3), and c-Myc pathways can induce the activity of HIF1 in a hypoxia independent manner[9]
Immunohistochemistry of bovine ovarian follicles revealed that hypoxia-inducible factor 1 A (HIF1A) proteins are distinctly expressed in the granulosa cells (GC) layer of primary, secondary, tertiary, and large antral follicles, which are in general under the influence of FSH and insulin-like growth factor 1 (IGF1) in-vivo (Fig. 1c)

Summary

Introduction

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor, consisting of a constitutively expressed β-subunit (HIF1B) and a regulated α-subunit (HIF1A). Our data suggest that CYP19A1, the key gene of estradiol production, is one of the plausible downstream targets of HIF1 in bovine GC as shown by gene expression, radioimmunoassay, and chromatin precipitation analysis Based on these results, we propose that HIF1 driven transcriptional activity plays a crucial role in GC functionality, especially steroidogenesis and proliferation in developing bovine ovarian follicles. Alam et al (2004) reported that FSH (follicle stimulating hormone) mediated www.nature.com/scientificreports upregulation of genes involved in follicle differentiation such as VEGFA (vascular endothelial growth factor A), LHCGR (Luteinizing Hormone/Choriogonadotropin Receptor) and INHBA (inhibin-α) is dependent on HIF1 activity in rats[16] Another gene, END2 (endothelin 2), which is suggested to play a role in ovulation and luteinization processes, was found to be regulated by HIF1 in transformed human GC17. The present investigation was carried out to identify HIF1 dependent transcriptional activity both under normoxic and hypoxic conditions using our renowned estrogen active culture model of bovine primary GC20–23

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