Human umbilical cord mesenchymal stem cells improve the ovarian function through oxidative stress-mediated PERK/eIF-2α/ATF4/CHOP signaling in premature ovarian insufficiency mice.

Abstract

Background: Premature ovarian insufficiency (POI) is a refractory disease that severely affects female fertility. The PERK/eIF-2α/ATF4/CHOP pathway is one of the classical pathways involved in the unfolded protein response to endoplasmic reticulum stress by regulating protein synthesis and promoting apoptosis. This study aimed to investigate the functional role and mechanism of human umbilical cord mesenchymal stem cells (hUCMSCs) in the POI animal model through the PERK/eIF-2α/ATF4/CHOP pathway.

Methods and results: Forty-five sexually mature female C57 mice were divided into a blank control group, POI model group, and hUCMSCs intervention group. To establish the POI model, mice received intraperitoneal injections of cyclophosphamide (CTX) (70 mg/kg) daily for 14 consecutive days, while the control group received saline only. In the hUCMSC intervention group, mice were given hUCMSCs on days 14 and 28, based on CTX modeling in the POI model group. The hUCMSCs were isolated, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) fluorescent dye, and tail vein-injected, and the distribution of the DiR signal was monitored in the mice using a fluorescence imaging detection method. The ovarian tissues were hematoxylin and eosin stained to observe the ovarian structure, and the number of primordial follicles were counted. An enzyme-linked immunosorbent assay was used to detect the serum levels of estradiol, anti-mullerian hormone, and follicle-stimulating hormone. Terminal deoxynucleotidyl transferase dUTP nick end labeling was used to detect the apoptosis of granulosa cells (GCs). The reactive oxygen species (ROS) content of ovarian tissue was detected by flow cytometry assay. The RNA expression of PERK, eIF-2α, ATF4, and CHOP was determined by quantitative real-time polymerase chain reaction, and protein levels of the targets were determined by western blot and immunohistochemistry. We identified hUCMSCs using surface antigenic markers (CD90, CD44, CD105, and CD73), and osteoblasts and chondroplast differentiation assays. Our studies demonstrated that hUCMSC intervention significantly restored ovarian function by improving the irregular estrous cycle, increasing the number of follicles, decreasing ROS, and inhibiting GC apoptosis in POI mice. Moreover, hUCMSCs suppressed CTX-induced PERK/eIF-2a/ATF4/CHOP pathway activation.

Conclusions: HUCMSCs can migrate to the damaged ovaries of POI mice, and improve the ovarian function of POI mice by inhibiting oxidative stress, down-regulating the expression of the PERK/eIF-2α/ATF4/CHOP pathway, and reducing the apoptosis of GCs.