MSCs-derived EVs protect against chemotherapy-induced ovarian toxicity: role of PI3K/AKT/mTOR axis

Abstract

Chemotherapy detrimentally impacts fertility via depletion of follicular reserves in the ovaries leading to ovarian failure (OF) and development of estrogen deficiency-related complications. The currently proposed options to preserve fertility such as Oocyte or ovarian cortex cryopreservation are faced with many technical obstacles that limit their effective implementation. Therefore, developing new modalities to protect ovarian function remains a pending target. Exosomes are nano-sized cell-derived extracellular vesicles (EVs) with documented efficacy in the field of regenerative medicine. The current study sought to determine the potential beneficial effects of mesenchymal stem cells (MSCs)-derived EVs in experimentally induced OF. Female albino rats were randomly allocated to four groups: control, OF group, OF + MSCs-EVs group, OF + Rapamycin (mTOR inhibitor) group, and OF + Quercetin (PI3K/AKT inhibitor) group. Follicular development was assessed via histopathological and immunohistochemical examination, and ovarian function was evaluated by hormonal assay. PI3K/Akt/mTOR signaling pathway as a key modulator of ovarian follicular activation was also assessed. MSCs-EVs administration to OF rats resulted in restored serum hormonal levels, preserved primordial follicles and oocytes, suppressed ovarian PI3K/AKT axis and downstream effectors (mTOR and FOXO3), modulated miRNA that target this axis, decreased expression of ovarian apoptotic markers (BAX, BCl2) and increased expression of proliferation marker Ki67. The present study validated the effectiveness of MSCs-EVs therapy in preventing ovarian insufficiency induced by chemotherapy. Concomitant MSCs-EVs treatment during chemotherapy could significantly preserve ovarian function and fertility by suppressing the PI3K/Akt axis, preventing follicular overactivation, maintaining normal ovarian cellular proliferation, and inhibiting granulosa cell apoptosis.

Keywords: Chemotherapy; Fertility preservation; MSCs-derived exosomes; Ovaries; PI3K/Akt/ mTOR.

Fig. 1

Schematic presentation of the timeline of treatments

Fig. 2

Representative photomicrographs of CD105 (MSCs marker) immuno-stained sections. The positive immunoexpression for CD105 was observed in MSCs-EVs treated group

Fig. 3

Representative photomicrographs of H&E-stained sections: a, b) Group I: Ovarian follicles (F) in various stages of development, and blood vessels (bv). c, d) Group II: extensive atresia of the follicles, with shrunken darkly stained nuclei (A), dilated congested blood vessels (bv), areas of hemorrhage (H), and degenerated follicles (D). e, f) Group III: Normal ovarian architecture, with developing follicles (F), blood vessels (bv), and corpus luteum (CL), g, h and i, j) Group IV and V: Fewer atretic follicles (A) in both groups with more numerous developing follicles (F) in Group IV. Persistent dilated congested blood vessels (bv) are noticed in both groups. k) Histogram demonstrating ovarian follicle count

Fig. 4

Effect of MSCs-EVs, rapamycin, and quercetin on (A) serum Estradiol (E2), (B) serum anti-mullerian hormone (AMH), (C), serum follicle stimulating hormone (FSH), (D) serum luteinizing hormone (LH). Results are expressed as mean ± SEM. *** significant Vs control group at p<0.001, **** at p<0.0001, # significant vs. OF group at p<0.05, ## at p<0.01, ### at p<0.001, #### at p<0.0001, & significant vs. OF + Rapamycin group at p<0.05, &&&& at p<0.0001, $$$$ significant vs. OF + quercetin group at p<0.0001

Fig. 5

Effect of MSCs-EVs, rapamycin, and quercetin on (A) relative gene expression of PTEN, (B) FOXO3, (C) Ovarian levels of PTEN and FOXO3 as detected by western blot analysis, (D, E) Intensity of immunoreactivity of selected proteins as quantified by densitometry. Results are expressed as mean ± SEM. *significant vs. Control group at p<0.05, ** at p<0.01, **** at p<0.0001, # significant vs. OF group at p<0.05, ## at p<0.01, ### at p<0.001, #### at p<0.0001. & significant vs. OF + Rapamycin group at p<0.05, $ significant vs. OF + quercetin group at p<0.05, $$ at p<0.01, $$$$ at p<0.0001

Fig. 6

Effect of MSCs-EVs, rapamycin, and quercetin on (A) phosphorylation of mTOR, PI3K, and AKT as detected by western blot analysis, (B-D) Intensity of immunoreactivity of selected proteins as quantified by densitometry. Results are expressed as mean ± SEM. **significant vs. Control group at p<0.01, **** at p<0.0001, # significant vs. OF group at p<0.05, ## at p<0.01, ### at p<0.001, #### at p<0.0001, & significant vs. OF + Rapamycin group at p<0.05

Fig. 7

Effect of MSCs-EVs, rapamycin, and quercetin on (A) miRNA-200c (B) miRNA-122 (C) miRNA-99a relative expression in ovarian tissues. Results are expressed as mean ± SEM. *significant vs. Control group at p<0.05, ** at p<0.01, **** at p<0.0001, #### significant vs. OF group at p<0.0001. &&&& significant vs. OF + Rapamycin group at p<0.0001, $$$$ significant vs. OF + quercetin group at p<0.0001

Fig. 8

A) Analysis of the biological pathways affected by PTEN, FOXO3 genes using FunRich software. P < 0.05 indicates a statistically significant difference. B) Molecular interaction network of PTEN, FOXO3 genes determined using FunRich software. P < 0.05 was considered to indicate a statistically significant difference. C) Biological pathways affected by miR-200c, miR-122, and miR-99 according to FunRich software. P < 0.05 indicates a statistically significant difference

Fig. 9

Representative photomicrographs of Ki67 immuno-stained sections. a, b) Group I: intense, c, d) Group II: weak, e, f) Group III: moderate, g, h) Group IV: mild, i, j) Group V: weak, nuclear immunoreaction in the granulosa cells of the follicles. k): Histogram demonstrating healthy and atretic follicles. l) Allred score for Ki67 immuno reactivity, * significant vs. Control group at p<0.05, *** at p<0.001, ## significant vs. OF group at p<0.01, #### at p<0.0001. $$ significant vs. OF + Quercetin group at p<0.01

Fig. 10

Representative photomicrographs of BAX immuno-stained sections. a, b) Group I: weak (curved arrow), c, d) Group II: intense, e, f) Group III: weak, g, h) Group IV: moderate, i, j) Group V: intense nuclear immunoreaction in the granulosa cells of the follicles. k) Histogram demonstrating healthy and atretic follicles. l) Allred score for BAX immunoreactivity, ****significant vs. Control group at p<0.0001, ## significant vs. OF group at p<0.01, #### at p<0.0001. &&&& significant vs. OF + Rapamycin group at p<0.0001, $$$$ significant vs. OF + Quercetin group at p<0.0001

Fig. 11

Representative photomicrographs of BCL2 immuno-stained sections. a, b) Group I: intense, c, d) Group II: weak, e, f) Group III: intense, g, h and i, j) Group IV & V: moderate and weak immunoreaction in the numerous and few granulosa cells respectively. k) Histogram demonstrating healthy and atretic follicles. l) Allred score for BCL2 immunoreactivity, **** significant vs. Control group at p<0.0001, #### significant vs. OF group at p<0.0001, & significant vs. OF + Rapamycin group at p<0.05, $$$$ significant vs. OF + Quercetin group at p<0.0001