P-610 Endoplasmic reticulum stress-induced Notch signaling stimulates cumulus-oocyte complex expansion in PCOS
Authors: Koike H , Harada M , Kunitomi C , Kusamoto A , Xu Z , Tanaka T , Urata Y , Osuga Y 
Published: June 2022

Abstract

Study question
Does endoplasmic reticulum (ER) stress and Notch signaling affect cumulus-oocyte complex (COC) expansion in pathophysiology of polycystic ovary syndrome (PCOS)?

Summary answer
Notch signaling is induced via activation of ER stress in granulosa cells (GCs) of PCOS and stimulates COC expansion that is abrogated by Notch inhibition.

What is known already
PCOS presents a variety of symptoms including ovarian dysfunction which is caused by various local factors in follicular microenvironment; among them, ER stress and following activation of unfolded protein response are critical, causing ovarian fibrosis, growth arrest of antral follicles and other ovarian dysfunctions. While Notch signaling pathway plays an important role of various ovarian functions such as ovarian development, follicle growth, luteinization and steroid hormone synthesis, the potential interaction between Notch signaling and ER stress in ovarian function is not determined.

Study design, size, duration
To examine expression levels of Notch signaling, ovaries and granulosa-lutein cells (GLCs) were collected from PCOS patients undergoing surgery or IVF. Human GLCs were collected from follicular fluid of IVF patients and cultured under ER-stressed condition. COCs obtained from PMSG-primed mice were subjected to examine the in vitro effects of ER stress activation and Notch inhibition on COC expansion. To examine the in vivo effects of Notch inhibition, dehydroepiandrosterone-induced PCOS mouse model was used.

Participants/materials, setting, methods
The expression levels of Notch signaling in ovaries and GLCs were investigated by immunohistochemistry and real time qPCR. To examine whether Notch signaling is activated by ER stress, human GLCs were incubated with ER stress inducer or inhibitor and ATF4 was knocked down by RNA interference. To investigate COC expansion level, murine COCs were cultured under ER stress condition with/without Notch signaling inhibitor. The COCs were collected from PCOS mice treated with/without Notch inhibitor.

Main results and the role of chance
We found that the expression levels of Notch2 and Hey2, a transcription factor activated by Notch signaling, were upregulated in GCs of antral follicles from PCOS patients and PCOS mice by using immunohistochemical analysis. Similarly, mRNA levels of these genes were higher in GLCs from PCOS patients than those from control patients. Notch signaling was induced in cultured human GLCs incubated with an ER stress inducer, tunicamycin; the effect was abrogated by incubation with an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), or knockdown of activating transcription factor 4 (ATF4, a transcription factor induced by ER stress). These findings suggest that Notch signaling is induced by ER stress via ATF4 pathway in human GCs. Measuring under a microscope, the area of expanded COCs was increased in cultured murine COCs incubated with tunicamycin, while this stimulatory effect of tunicamycin was abrogated by adding a Notch signaling inhibitor, DAPT. The area of expanded COCs obtained from PCOS model mice was increased compared to control mice, while administration of DAPT to these mice reduced the area. These results suggest that ER stress-induced Notch signaling stimulate COC expansion contributing PCOS pathophysiology.

Limitations, reasons for caution
COC expansion area was measured only in PCOS model mouse; it is unknown whether COC expansion is induced in PCOS patients. This point requires further investigation in PCOS patients.

Wider implications of the findings
Our findings suggest that ER stress-induced Notch signaling affects COC expansion, associated with ovulatory dysfunction in PCOS. The detailed understandings of PCOS pathophysiology may be beneficial for substantial clinical implications and inhibition of ER stress or Notch signaling may serve as a novel therapeutic approach for PCOS.

Trial registration number
This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (19k09749, 19k24045, 19k24021, 21k16808, 21j12871,), a grant from the Takeda Science Foundation, a grant from The Tokyo Society of Medical Science, a grant from The Japan Society of Fertility Preservation, and a grant from The Japan Society for Menopause and Women’s Health (JMWH) (a JMWH Bayer Grant).