HIF-1α Protects Granulosa Cells From Hypoxia-Induced Apoptosis During Follicular Development by Inducing Autophagy.

Zonghao Tang,Fengping Lin,Yiping Liu,Zhengchao Wang,Baorong Geng,Congjian Shi,Renfeng Xu,Qingqiang Lin,Zhenghong Zhang,Yan Zhang,Hongqin Yang

doi:10.3389/fcell.2021.631016

Abstract

Owing to the avascular structure of the ovarian follicle, proliferation of granulosa cells (GCs) and development of follicles occur under hypoxia, which is obviously different from the cell survival requirements of most mammalian cells. We hypothesized that autophagy may exert an inhibitory effect on GC apoptosis. To decipher the underlying mechanism, we constructed a rat follicular development model using pregnant mare serum gonadotropin and a cell culture experiment in hypoxic conditions (3% O2). The present results showed that the autophagy level was obviously increased and was accompanied by the concomitant elevation of hypoxia inducible factor (HIF)-1α and BNIP3 (Bcl-2/adenovirus E1B 19kDa-interacting protein 3) in GCs during follicular development. The levels of Bax (Bcl2-associated X) and Bcl-2 (B-cell lymphoma-2) were increased, while the activation of caspase-3 exhibited no obvious changes during follicular development. However, inhibition of HIF-1α attenuated the increase in Bcl-2 and promoted the increase in Bax and cleaved caspase-3. Furthermore, we observed the downregulation of BNIP3 and the decrease in autophagy after treatment with a specific HIF-1α activity inhibitor (echinomycin), indicating that HIF-1α/BNIP3 was involved in autophagy regulation in GCs in vivo. In an in vitro study, we also found that hypoxia did not obviously promote GC apoptosis, while it significantly enhanced the activation of HIF-1α/BNIP3 and the induction of autophagy. Expectedly, this effect could be reversed by 3-methyladenine (3-MA) treatment. Taken together, these findings demonstrated that hypoxia drives the activation of HIF-1α/BNIP3 signaling, which induces an increase in autophagy, protecting GC from apoptosis during follicular development.

Highlights

Oxygen is one of the major factors influencing the survival of mammalian cells, and the maintenance of oxygen tension within a physiological range is critical to ensure proper cell metabolism required for cellular functions (Zhang et al, 2011a,b, 2019a; Tang et al, 2017a, 2019)
Previous investigations have demonstrated an increase in the autophagy level in rat granulosa cells (GCs) (Choi et al, 2010), and that the accumulation of autophagosomes is an important factor leading to GC apoptosis (Choi et al, 2011)
The results showed that autophagy marker protein LC-3I/II was mainly expressed in GCs and increased either 6 or 12 h after treatment (Figure 1)

Summary

Introduction

Oxygen is one of the major factors influencing the survival of mammalian cells, and the maintenance of oxygen tension within a physiological range is critical to ensure proper cell metabolism required for cellular functions (Zhang et al, 2011a,b, 2019a; Tang et al, 2017a, 2019). In order to maintain normal supply of oxygen and nutrients, the developing tissues are ubiquitously accompanied by invasion of blood vessels. The proliferation of GCs and the expansion of follicles further exacerbate the imbalance of oxygen supply and consumption in ovarian follicles during follicular development (Fischer et al, 1992; Wang et al, 2012, 2015, 2017; Zhang et al, 2012; Wu et al, 2015, 2016). It has been demonstrated that the oxygen tension of follicular fluid in a small follicle is higher than that of large follicles and exhibits a lower trend during follicular maturation (Fischer et al, 1992; Zhang et al, 2012). The reason why GCs still maintain its proliferative phenotype under hypoxia conditions remains poorly understood

Methods

Results

Conclusion