Extracellular Vesicles Derived from Mesenchymal Stem Cells Recover Fertility of Premature Ovarian Insufficiency Mice and the Effects on their Offspring

Abstract

It has been reported that extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (HUCMSCs) can promote the proliferative and secretive functions of granulosa cells. In vivo study further demonstrated that EVs derived from HUCMSCs can not only promote the angiogenesis of ovarian tissue but also restore the function of an ovary of chemically induced premature ovarian insufficiency (POI) mice. However, no study investigates the effects of HUCMSCs derived EVs on fertility recovery of POI mice and evaluating their offspring. This study investigates the effects of HUCMSCs derived EVs on fertility recovery and the cognitive function of their offspring. A POI model was established by intraperitoneal injection of cyclophosphamide (CTX) and busulfan (BUS), and randomly divided into EVs-transplantation group (a single injection of 150 µg EVs proteins which suspended in 0.1 ml phosphate buffer saline [PBS] via tail vein), POI group (a single injection of 0.1 ml PBS via tail vein), and normal control group (a single injection of 0.1 ml PBS via tail vein without intraperitoneal injection of CTX and BUS). After EVs treatment, not only the ovarian function of POI mice recovered but also the fertility increased with less time to get pregnant, evaluating by in vitro fertilization and mating test. Cognitive behaviors of the offspring were similar among the three groups through the Y-maze test and novel object recognition task. An anti-apoptotic effect was identified through immunohistochemistry, real-time polymerase chain reaction and western blot. These findings indicate that HUCMSCs derived EVs can improve the fertility of POI mice without adverse effects on the cognitive behavior of their offspring, highlighting the potential value of EVs to be a cell-free therapy for patients suffering from POI.

Keywords: extracellular vesicles; fertility; in vitro fertilization; mesenchymal stem cell; offspring; premature ovarian insufficiency.

Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Fig. 1.

Characterizations of HUCMSCs and EVs. (A) Fibroblast-like morphology of the sixth passage HUCMSCs. (B) The protein expressions of CD9, CD63, TSG101, and CANX in the EVs and HUCMSCs by western blot. (C–D) The transmission electron microscope images of EVs. HUCMSCs: human umbilical cord mesenchymal stem cells; EVs: extracellular vesicles.

Fig. 2.

Schematic diagram of the experimental protocol. CTX: cyclophosphamide; BUS: busulfan; EVs: extracellular vesicles; IVF: in vitro fertilization.

Fig. 3.

Evaluation of the POI model. (A) The body weight of mice in the NC and POI groups (n = 10, *P < 0.05). (B) The concentrations of E2 in the NC and POI groups (n = 6). (C) The concentrations of FSH in the NC and POI groups (n = 6). (D) Histopathologic images of ovaries in the NC group (a) and POI groups (b). (E) The number of follicles at different development stages. Data represent means ± SEM, n = 6; *P < 0.05. (F) Representative photograph of ovaries in the NC and POI group. (G) The weight of ovaries in the NC and POI groups (n = 6). NC: normal control; POI: premature ovarian insufficiency; FSH: follicle-stimulating hormone.

Fig. 4.

EVs treatment improves the ovarian function of POI mice. (A) Body weight in 3 groups during the experimental period (n = 10, *P < 0.05 vs. NC; #P < 0.05 vs. POI). (B) E2 concentrations in the NC, POI and EVs groups during the experimental period (n = 6, *P < 0.05 vs. NC; #P< 0.05 vs. POI). (C) FSH concentrations in the NC, POI and EVs groups during the experimental period (n = 6, *P < 0.05 vs. NC; #P < 0.05 vs. POI). (D) Histopathologic images of ovarian tissue 28 days after interventions in the NC (a), POI (b), and EVs (c) groups. (E) The follicles of different development stages in three groups (n = 6). (F) Ovarian weight in three groups during the experimental period (n = 6, *P < 0.05 vs. NC; #P < 0.05 vs. POI). NC: normal control; POI: premature ovarian insufficiency; EVs: extracellular vesicles; FSH: follicle-stimulating hormone.

Fig. 5.

EVs transplantation improves the outcomes of in vitro fertilization and natural fertility of POI mice with no adverse effects on their offspring. (A) The numbers of oocytes retrieved, fertilized zygotes, cleaved embryos, and blastocysts in the NC, POI, and EVs groups (n = 6). (B) The time to get pregnancy in three groups evaluated by Log-rank (Mantel-Cox) test (P < 0.05 vs. POI, n = 15). (C) The average numbers of offspring in the NC, POI, and EVs groups (*P < 0.05 vs. NC; #P < 0.05 vs. POI). (D) The body weight of offspring in three groups (n = 25). (E, F) The Y-maze test and NORT results of offspring in three groups (n = 12). NC: normal control; POI: premature ovarian insufficiency; EVs: extracellular vesicles; NORT: novel object recognition task.

Fig. 6.

Anti-apoptotic effect of EVs. (A–C) The cleaved caspase 3 positive cells in ovarian tissue of the NC (A), POI (B), and EVs (C) group (n = 6). The mRNA expressions of caspase 3 (D), Bcl-2 (E), Bax (F), and Bcl-2/Bax (G) in ovarian tissue of three groups 28 days after interventions. Data represent means ± SEM; n = 7, *P < 0.05 vs. NC. (H) Western blotting detected protein expressions of caspase 3 (I), Bcl-2 (J) and Bax (K) in ovarian tissue of three groups 28 days after interventions. Data represent means ± SEM; n = 7, *P < 0.05 vs. NC; #P < 0.05 vs. POI. NC: normal control; POI: premature ovarian insufficiency; EVs: extracellular vesicles; mRNA: microribonucleic acid.