Evidence of the activation of unfolded protein response in granulosa and cumulus cells during follicular growth and maturation

Abstract

The objective of the present study is to investigate whether unfolded protein response (UPR), activated by endoplasmic reticulum (ER) stress, in granulosa cells (GC) and cumulus cells (CC) is involved in the process of follicular growth and maturation. First, to examine the presence of UPR in growing follicles, the expression of spliced form of X-box-binding protein 1 (XBP1(S)) and heat shock 70 kDa protein 5 (HSPA5) mRNA, typical UPR genes, in mice ovaries were examined by in situ hybridization. GC of later stage than large secondary follicles expressed both XBP1(S) and HSPA5 mRNA, which was accompanied with the activation of ER stress sensor proteins, inositol-requiring enzyme 1 (IRE1) and double-stranded RNA-activated protein kinase-like ER kinase (PERK), confirmed by immunohistochemistry. Next, to examine the association between the fertilization capacity of oocytes and the expression levels of UPR genes in surrounding CC, human CC were collected from patients undergoing ICSI. The expression levels of XBP1(S) mRNA, quantified by RT-PCR, in CC enclosing human oocytes achieving fertilization were two-fold higher, than those in CC enclosing oocytes without fertilization capacity. In conclusion, UPR is activated during follicular growth and suggested to be involved in the process of follicular growth and maturation.

Chinese abstract

本研究目的在于探索由内质网（endoplasmic reticulum，ER）应激、颗粒细胞（granulosa cells，GC）及卵丘细胞（cumulus cells ，CC）激活的非折叠蛋白应答（unfolded protein response，UPR）是否与卵泡的发育与成熟过程相关。首先，为了检测UPR在生长卵泡中的发生情况，研究测定了X盒结合蛋白1迭接体（spliced form of X-box-binding protein 1，XBP1(S)）、热休克70 kDa蛋白5（heat shock 70 kDa protein 5 (HSPA5)）mRNA及典型UPR基因，在小鼠卵巢应用原位杂交技术进行测定。免疫组化法证实，在次级卵泡之后的生长阶段，GC表达XBP1(S)与HSPA5 mRNA，并伴随ER应激传感蛋白、肌醇依赖酶1（inositol-requiring enzyme 1(IRE1)）与双链RNA激活蛋白激酶样ER激酶（PERK）的激活。然后，为检测卵母细胞的受精能力与周围卵丘细胞中UPR基因表达情况间的相关性，研究收集了进行ICSI治疗患者的卵丘细胞。经RT-PCR技术定量测定XBP1(S) mRNA的表达情况，其在受精的卵母细胞周围颗粒细胞中的表达是无受精能力卵母细胞周围颗粒细胞的2倍。综上，UPR在卵泡的发育阶段被激活，可能与卵泡发育及成熟的过程相关。

• Cumulus cells
• endoplasmic reticulum stress
• follicular growth and maturation
• granulosa cells
• unfolded protein response

Acknowledgements

We thank Dr. K. M. J. Menon of Departments of Obstetrics/Gynecology and Biological Chemistry, University of Michigan Medical School for his valuable comments.

Declaration of interest

The authors report no conflicts of interest. This work was supported by Grant-in-Aids for Scientific Research from Japan Society for the Promotion of Science.