Therapeutic effects of human umbilical cord mesenchymal stem cell-derived extracellular vesicles on ovarian functions through the PI3K/Akt cascade in mice with premature ovarian failure

Abstract

Premature ovarian failure (POF) mainly refers to ovarian dysfunction in females younger than forty. Mesenchymal stem cells (MSCs) are considered an increasingly promising therapy for POF. This study intended to uncover the therapeutic effects of human umbilical cord MSC-derived extracellular vesicles (hucMSCEVs) on POF. hucMSCs were identified by observing morphology and examining differentiation capabilities. EVs were extracted from hucMSCs and later identified utilizing nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. POF mouse models were established by injecting D-galactose (Dgal). The estrous cycles were assessed through vaginal cytology, and serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), anti-mullerian hormone (AMH), estradiol (E2), and progesterone (P) were measured by ELISA. The human ovarian granulosa cell line KGN was used for in vitro experiments. The uptake of hucMSC-EVs by KGN cells was detected. After D-gal treatment, cell proliferation and apoptosis were assessed via CCK-8 assay and flow cytometry. The PI3K/Akt pathway-related proteins were determined by Western blotting. Our results revealed that POF mice had prolonged estrous cycles, increased FSH and LH levels, and decreased AMH, E2, and P levels. Treatment with hucMSC-EVs partially counteracted the above changes. D-gal treatment reduced proliferation and raised apoptosis in KGN cells, while hucMSC-EV treatment annulled the changes. D-gal-treated cells exhibited downregulated p-PI3K/PI3K and p-Akt/Akt levels, while hucMSC-EVs activated the PI3K/Akt pathway. LY294002 suppressed the roles of hucMSC-EVs in promoting KGN cell proliferation and lowering apoptosis. Collectively, hucMSC-EVs facilitate proliferation and suppress apoptosis of ovarian granulosa cells by activating the PI3K/Akt pathway, thereby alleviating POF.

Introduction

Premature ovarian failure (POF), also known as primary or premature ovarian insufficiency (POI), is an infertility disorder in humans that affects 1-5% of women younger than 40 years.¹ The occurrence of POF is mainly attributed to dysfunctional ovarian follicles or decreased primordial follicles.² The nongenetic causes of POF include autoimmune diseases, viral infections, radiotherapy, or chemotherapy, while genetic causes are highly heterogeneous, including both isolated (nonsyndromic) and syndromic POF.³ The typical symptoms of POF consist of hormonal deficits, infertility, vaginal dryness, discomfort during sexual intercourse, irregular menstruation, night sweating, and emotional problems.⁴ In terms of treatments, in vitro activation, regenerative medicine, and hormone therapy are well-known and commonly utilized therapies for POF; however, these modalities may be accompanied by several complications.^5,6 Thereby, more effective and potent therapeutic strategies for POF are of utmost urgency and importance to restore ovarian function, thus alleviating adverse consequences.

Extracellular vesicles (EVs) are intrinsically considered to be membrane-bound vesicles released from prokaryotic and eukaryotic cells, which can potentially transport multifarious cargoes including DNA, RNA, proteins, and lipids, serving as vital vehicles of intercellular communication.^7,8 Based on the distinct biological properties, including size, biomarker, and biogenesis pathways, EVs are classified mainly into apoptotic bodies, microvesicles, and exosomes.⁹ Typically, an array of cells, such as cancer cells, endothelial cells, epithelial cells, platelets, leukocytes, neurons, fibroblasts, and mesenchymal stem cells (MSCs), can secrete EVs.^10,11 Due to the self-renewal and regeneration potential,¹² stem cells are exceedingly utilized to potently treat POF, with beneficial effects on ovarian angiogenesis, structural and functional restoration, follicular activation, and hormonal level recovery.^4,13 As one of the intensively studied MSCs, human umbilical cord MSCs (hucMSCs) emerge as ideal therapeutic means due to their unique advantages such as large cell content, low cost, noninvasive collection procedures, easy extraction and expansion, low immunogenicity, high proliferation, and low infection risk, and the hucMSCderived EVs (hucMSC-EVs) have shown therapeutic effects in cardiovascular, neurological, kidney, liver, and bone diseases, cancer, and infertility.¹⁴ To this end, investigating the precise therapeutic roles of hucMSC-EVs in POF is imperative for clinical treatment.

Many researchers have documented the paramount importance of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) cascade in primordial follicle activation.^15,16 The suppressed PI3K/Akt signaling essentially prevents granulosa cell growth and induces apoptosis,¹⁷ which also probably contributes to large-scale oocyte loss, ultimately causing POF.¹⁸ Interestingly, Evodiae fructus extract alleviates 4-vinylcyclohexene diepoxide-induced ovotoxicity in ovary cells by activating the PI3K/Akt pathway, showing potential in preventing POF.¹⁹ Therefore, we proposed a hypothesis that MSC-EVs might exert a therapeutic role in POF through the PI3K/Akt pathway. Accumulating reports reveal that D-galactose (D-gal) treatment leads to excessive formation of reactive oxygen species (ROS) and accumulation of advanced glycation endproducts (AGE) in the brain, myocardium, liver, kidney, muscle, blood vessel, and ovary of mice,^20-2, which are primary causes of ovarian aging that gradually impairs ovarian functions.²⁶ Because the accelerated aging process in the D-gal-induced aging model is highly similar to that occurring during aging in humans, this model is widely used to explore the mechanism of POF.². Therefore, in the current study, POF mouse models were established by injecting D-gal to explore the action and mechanism of hucMSC-EVs in POF, so as to provide new insights into POF treatment and management.

Materials and Methods

Culture and identification of hucMSCs

The hucMSC line CP-CL11 provided by Procell (Wuhan, China) was cultured in Dulbecco’s modified Eagle medium (DMEM) added with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100% μg/mL streptomycin (Solarbio, Beijing, China) in a 37°C incubator with 5% CO².

The procedures of Oil red O staining for detection of adipogenic differentiation of hucMSCs were as follows: hucMSCs were induced for adipogenic differentiation using OriCell^TM hucMSC Adipogenic Differentiation Kits (HUXUC-90031, Cyagen Biosciences, Santa Clara, CA, USA). Cells were seeded at a density of 2×10⁴ cells/cm² in 6-well culture plates and subsequently cultured in human MSC serum-free medium. When confluence reached 100%, cells were cultured in hucMSC adipogenic differentiation medium for 21 days and then fixed with 4% neutral formaldehyde for 30 min at room temperature. Cells were stained with 0.5% Oil red O (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for 40 min at room temperature. Cells were rinsed twice with phosphate-buffered saline (PBS), stained with Harris hematoxylin (Surgipath, Bretton, UK) for 45 s, and washed with tap water (turned blue) for 1 to 3 min, followed by washing with distilled water. The peripheral water was aspirated using paper towels, and the slides were mounted using glycerin gelatin. Cells were observed under a light microscope (Olympus, Tokyo, Japan) at a magnification of 200× and images were captured.

The steps of Alizarin red staining for detection of osteogenic differentiation of hucMSCs were as follows: osteogenic differentiation was induced in hucMSCs by OriCell^TM hucMSC Osteogenic Differentiation Kits (HUXUC-90021, Cyagen Biosciences). Cells were seeded at a density of 1×10⁴ cells/cm² in 24-well culture plates and subsequently cultured in human MSC serum-free medium. When confluence reached 80-90%, cells were cultured in hucMSC osteogenic differentiation medium for 21 days and subsequently fixed for 30 min with 4% neutral formaldehyde at room temperature. Cells were subsequently washed with PBS for 2 min, stained with 10% Alizarin red (Yeasen, Shanghai, China) for 10 min at room temperature, and rinsed thrice with PBS. The fluid was discarded, and the slides were mounted with neutral gum. Cells were observed under a light microscope (200×, Olympus) and images were obtained.

EVs extraction and identification

The conditional medium (CM) of hucMSCs was centrifuged at 2,000×g for 30 min at 4°C to remove cell debris, and next the supernatant was ultracentrifuged (Optima XPN-100, Beckman Coulter, Chaska, MN, USA) at 100,000×g for 2 h at 4°C. The supernatant was discarded, and the sediments were resuspended in PBS and ultracentrifuged again at 100,000×g for 2 h. The concentrated hucMSC-EVs were then resuspended in PBS.²⁸ The number of hucMSC-EVs was determined by measuring total protein content using the Bradford method, and the diluted bovine serum albumin gradient was employed to plot standard curves.^29,30 The morphology of EVs was observed under a transmission electron microscope (TEM; HT7800, Hitachi, Japan), and the particle size was measured with a nanoparticle tracking analyzer (NTA; Malvern Instruments, Malvern, UK). GW4869 is widely used to inhibit the secretion of EVs,^31,32 and therefore we used it as a negative control for EVs in this study. hucMSCs were cultured in EV-free FBS medium supplemented with GW4869 (20 μg/mL CM; Sigma- Aldrich) for 48 h and later the CM was taken and used as the control. The expression levels of EV surface markers Calnexin (ab22595, Abcam, Cambridge, UK), CD9 (ab236630, Abcam), CD81 (ab219209, Abcam), and CD63 (ab134045, Abcam) were determined using Western blotting.

Experimental animals

C57BL/6 female mice aged 6-8 weeks were acquired from SIMR Biotechnology (License No. SCXK [Shanghai] 2022-0004, Shanghai, China). Mice were housed in a pathogen-free animal facility under 12-h dark/light cycles, with ad libitum access to food and water. The D-gal-induced POF model is used as a model of ovarian aging in mice.^33,34 Firstly, mice were randomized to 4 groups (6 mice per group): control group, POF group, POF + GW4869 group, and POF + EVs group. Mice in the POF, POF + GW4869, and POF + EVs groups were subcutaneously injected with 600 mg/kg/day D-gal (C₆H₁₂O₆, Sigma-Aldrich) once a day for 42 days. The control mice were only injected with an equal amount of normal saline. Mice in the POF + EVs/GW4869 groups were injected with D-gal and next injected with 0.2 mL PBS containing 1×10⁸/mL hucMSC-EVs or GW4869 through tail veins every 2 days. One week after the last injection, mice were euthanized with 800 mg/kg pentobarbital sodium and the peripheral blood of mice was collected.

Estrous cycles

Estrous cycles were monitored by vaginal smears for 12 consecutive days following D-gal treatment to assess the estrous cycle stages in mice. Vaginal lavage samples were diluted with sodium chloride injection at appropriate concentrations. Afterward, cells were evenly distributed over clean slides utilizing a centrifugal smear dyeing machine (Cytopro 7, 621, Norwalk, CA, USA), fixed in absolute ethanol for 10 s, and then stained with Wright-Giemsa Stain (Baso Diagnostics, Zhuhai, China). Vaginal cells were analyzed under a light microscope based on morphological criteria. The length of each cycle was evaluated as the time span between two consecutive estrous cycles.

ELISA

The peripheral blood samples were allowed to clot for 60 min and later centrifuged at 1,000×g for 15 min to collect the serum, followed by preservation at -80°C. The levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), anti-Mullerian hormone (AMH), estradiol (E2), and progesterone (P) were determined using ELISA kits.

Culture of ovarian granulosa cells

The ovarian granulosa cells (KGN cell line; Procell) were cultured in DMEM (Sigma-Aldrich) added with 10% FBS (Sigma- Aldrich) and 1% antibiotic/antifungal solution (Invitrogen, Carlsbad, CA, USA). KGN cells in the 2^nd generation were put in 6-well plates at a density of 1×10⁵ cells/well. After 24 h, the original medium was discarded, and cells were only treated with DMEM/F12 medium (Invitrogen) for 24 h to starve the cells. Thereafter, KGN cells were assigned into 6 groups: blank (without treatment), D-gal (medium was added with 100 nM D-gal³⁵), D-gal + EVs (medium was supplemented with 100 nM D-gal and hucMSC-EVs at a density of 1×10⁸ particles/mL), D-gal + GW4869 (the CM was added with 100 nM D-gal and equivalent GW4869), D-gal + EVs + LY294002 (medium was supplemented with 100 nM D-gal, an equivalent volume of hucMSC-EVs, and 50 μM PI3K/Akt inhibitor LY294002³⁶ purchased from Abcam), and D-gal + EVs + DMSO (medium was supplemented with 100 nM D-gal and equivalent volumes of hucMSC-EVs and dimethyl sulphoxide [DMSO]).

Labeling of EVs

The 100 μL of EV suspension was supplemented with 1 mL of dilution C-diluted PKH67 (Sigma-Aldrich) for 4 min. The reaction was subsequently terminated via exposure to 1 mL of 0.5% bovine serum albumin, and later the extraction kits were employed to reextract EVs. After labeling with PKH67, under a confocal laser scanning microscope EVs appeared green. KGN cells were stained blue with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). PKH67-labeled EVs were co-cultured with KGN cells for 24 h, followed by localization. The specific steps were as follows: KGN cells were cultured in 4-well plates for 24 h and then the well was added with PKH67-labeled hucMSC-EVs, followed by incubation for 24 h at 37°C. Thereafter, cells were rinsed thrice with cold PBS and fixed with 2% paraformaldehyde for 10 min at room temperature. Next, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 2 min, rinsed with PBS thrice, and stained with DAPI (10 μg/mL) for 5 min at room temperature. Images were then captured with a fluorescence microscope (Olympus).

CCK-8 assay

Cells were seeded in 96-well plates at a density of 2×10⁴ cells/mL. After 24 h, cell viability was examined by CCK-8 assay. In brief, the corresponding volume of CCK-8 reagent was added to each well and mixed well, followed by culture for 1.5 h in an incubator containing 5% CO² at 37°C. The optical density (OD) value at 450 nm was then determined on a microplate reader.

Flow cytometry

Cells were put in 24-well plates at a density of 1×10⁶ cells/mL. After 48-h incubation, cells were collected and resuspended. Afterward, 100 μL of cell resuspension was stained with 5 μL of 1 mg/mL Annexin V-fluorescein isothiocyanate and 5 μL of 2.5 mg/mL propidium iodide (Vazyme, Nanjing, China) for 20 min. Finally, the apoptosis rate was calculated utilizing a flow cytometer (BD Biosciences, San Jose, CA, USA). Each experiment was repeated thrice.

Western blotting

Protein was obtained through lysis of cells or EVs using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was measured with bicinchoninic acid kits (Sigma-Aldrich). Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by membrane transferring. Membranes were probed with 4 mL primary antibodies overnight at 4°C. Subsequently, membranes were rinsed thrice and later incubated with horseradish peroxidase- conjugated goat anti-rabbit secondary antibody for 2 h. The ratio of the gray value of the target protein bands to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reflected the relative expression of target proteins, and the relative protein expression was semi-quantitatively analyzed with image analysis software BandScan 5.0 (BioMarin, San Rafael, CA, USA). (Primary antibodies: Calnexin, 1:1000, ab22595; CD9, 1:1000, ab236630; CD81, 1:1000, ab219209; CD63, 1:1000, ab134045; PI3K, 1:1000, ab32089; p-PI3K, 1:1000, ab182651; Akt: 1:1000, ab38449; p-Akt: 1:1000, ab81283; GAPDH, 1:1000, ab9485; secondary antibody IgG: 1:2000, ab205718; all from Abcam).

Statistical analysis

Data analysis was processed using SPSS 21.0 (IBM Corp., Armonk, NY, USA) software. Measurement data were displayed as mean ± standard deviation (SD). The cell experiment was independently repeated thrice and the animal experiment was performed using 6 mice per group. The results of animal experiments were represented as scatter charts. The independent sample t-test was conducted for comparisons between two groups and one-way analysis of variance (ANOVA) was implemented for comparisons among multiple groups. Tukey’s multiple comparisons test was employed for the post hoc analysis. The p<0.05 was regarded as statistically significant.

Results

Identification of hucMSCs and EVs

To investigate the role of hucMSC-EVs, we purchased and then identified hucMSCs. Firstly, the morphology of hucMSCs was observed under a microscope, which indicated that hucMSCs grew in a monolayer mode and showed fibroblast-like characteristics (with a long-fusiform shape) (Figure 1A). Oil red O and Alizarin red stainings suggested that hucMSCs could differentiate into adipocytes or osteocytes after induction. After induction of adipogenic differentiation, a large number of long spindle-shaped or polygonal lipid droplets appeared in the cells; after induction of osteogenic differentiation, the whole cells were filled with calcium granules, the cells grew in colonies, and the central cells gradually fused, lost the typical cell structure, and formed clear calcium nodules (Figure 1B). In addition, the morphology and particle size of EVs were analyzed by TEM and NTA, which delineated that EVs appeared to be round or elliptical, with a complete bilayer vesicle structure and a diameter of 30-200 nm, which were in line with the general characteristics of EVs (Figure 1 C,D). Finally, Western blotting discovered that EV markers CD9, CD81, and CD63 were positively expressed, while the negative control Calnexin, an endoplasmic reticulum marker, was not expressed (Figure 1E). The aforementioned results elicited the successful isolation of hucMSC-EVs.

hucMSC-EVs ameliorated POF in mice

The mouse models of POF were induced using D-gal to validate whether hucMSC-EVs can affect POF. The observation through vaginal smears unveiled that D-gal-induced POF mice exhibited prolonged estrous cycles (Figure 2A). ELISA revealed that POF mice had increased FSH and LH levels, but decreased AMH, E2, and P levels in the serum (Figure 2B, all p<0.01). However, treatment with hucMSC-EVs partially annulled the above changes in estrous cycles and levels of serum hormones FSH, LH, AMH, E2, and P (Figure 2A-B, all p<0.01). Taken together, hucMSC-EVs could mitigate POF in mice to a significant extent.

hucMSC-EVs facilitated proliferation and prevented apoptosis of ovarian granulosa cells

To further explore whether hucMSC-EVs alleviate POF by mediating ovarian granulosa cells, the purchased ovarian granulosa cell line KGN was co-cultured with PKH67-labeled hucMSCEVs for 24 h. The uptake assay of hucMSC-EVs by KGN cells revealed that an enormous number of hucMSC-EVs entered KGN cells and distributed around the nucleus (Figure 3A). Subsequently, CCK-8 assay and flow cytometry delineated that Dgal- treated KGN cells showed lowered proliferation and raised apoptosis, while further hucMSC-EVs treatment to some extent ameliorated the decrease in proliferation and increase in apoptosis (Figure 3 B,C, all p<0.01). In summary, hucMSC-EVs potentiated proliferation and restrained apoptosis of ovarian granulosa cells.

Figure 1.

Identification of hucMSCs and EVs. A) Cell morphology of hucMSCs observed under an inverted microscope. B) Adipogenic and osteogenic differentiation abilities of hucMSCs assessed by oil red O and Alizarin red stainings, respectively. C) Ultrastructure of EVs observed through TEM; D) Diameter distribution and concentration of EVs detected by NTA. E) Expression of EV marker proteins CD9, CD81, CD63, and endoplasmic reticulum marker Calnexin determined by Western blotting. Cell experiment was independently repeated thrice.

hucMSC-EVs promoted functional recovery of ovarian granulosa cells through the PI3K/Akt pathway

Mounting evidence reveals the protection role of the PI3K/Akt pathway activation in ovarian aging,^37,38 and hence we proposed that hucMSC-EVs promote the functional restoration of ovarian granulosa cells by activating the PI3K/Akt pathway. The marker proteins of the PI3K/Akt pathway were determined by Western blotting, which unraveled that p-PI3K/PI3K and p-Akt/Akt in KGN cells were prominently downregulated after D-gal treatment; the downregulation of p-PI3K/PI3K and p-Akt/Akt was partially annulled upon hucMSC-EV treatment; further treatment with LY294002, a PI3K pathway inhibitor, weakened the effect of hucMSC-EVs on activating the PI3K/Akt pathway (Figure 4A, all p<0.01). Meanwhile, CCK-8 and flow cytometry demonstrated that LY294002 suppressed the roles of hucMSC-EVs in promoting KGN cell proliferation and lowering apoptosis rate (Figure 4 B,C, all p<0.01). Briefly, hucMSC-EVs promoted proliferation and repressed apoptosis of ovarian granulosa cells by activating the PI3K/Akt pathway, thereby improving POF.

Discussion

POF is commonly construed as a syndrome comprising sex steroid deficiency, gonadotropin elevation, and amenorrhea in women under the age of 40, resulting in infertility and considerable psychological stress.³⁹ EVs are currently gaining a great deal of attention from researchers for their specific release mechanisms, participation in cell-to-cell signaling, and utility as biomarkers or therapeutic targets for diseases.⁴⁰ Of note, MSC-EVs confer therapeutic roles in diverse female reproductive disorders, such as POI, polycystic ovary syndrome, and intrauterine adhesion.^41,42 Currently, studies on the treatment of POF are mostly based on in vitro cell experiments and in vivo animal models.^36,43-45 Prior clinical study has investigated the therapeutic use of intramuscular injection of MSCs for the treatment of uterine niche after cesarean delivery.⁴⁶ However, research on the therapeutic effect of hucMSCEVs on POF at the clinical level is indeed rare. This study explored the therapeutic mechanism of hucMSC-EVs in POF and highlighted that hucMSC-EVs restored ovarian function via the PI3K/Akt cascade in POF mice.

As a widely used model of aging in mice, the D-gal-induced POF model is generally employed to explore the potential mechanisms of ovarian aging, as accelerated aging extremely resembles the observation in humans.⁴⁷ Therefore, in our experiments, POF mouse models were also induced by subcutaneously injecting Dgal. It is broadly well-known that estrous cycles and sex hormone secretion are commonly used for the evaluation of ovarian function. ⁴⁸ POF is typically characterized by low AMH and gonadal hormones (inhibins and estrogens), high gonadotropins including FSH and LH (hypergonadotropic amenorrhea), and LH in conjunction with FSH can modulate the production of steroid sex hormones E2 and P through theca cells surrounding growing follicles in the ovary.^49,50 Not surprisingly, our results unearthed that POF mice presented prolonged estrous cycles, raised FSH and LH levels, and diminished AMH, E2, and p-levels in the serum. GW4869 (a cell-permeable symmetrical dihydroimidazolamide compound), is a potent, specific, and non-competitive inhibitor of membraneneutral sphingomyelinase,^51,52 which can efficiently inhibit the secretion of EVs.^31,32 Therefore, GW4869 was used as a negative control for EVs in this study. In subsequent experiments, hucMSCEV treatment shortened estrous cycles and partially reversed the above trends in serum hormones. Consistently, embryonic stem cell-secreted EVs can restore E2, AMH, and FSH to normal levels, reduce atresia, and elevate the number of normal follicles.³⁶ It is noteworthy that hucMSCs have the advantages of easy extraction and expansion, cost-effectiveness, non-invasive collection process, high cell content, and a small risk of infection, making them highly suitable for therapeutic applications.^14,53,54 As one of the major extracellular signaling pathways, EVs are associated with a variety of diseases and are involved in diverse physiological processes.^55,56 Compared to other EVs, hucMSC-EVs have a greater potential for treatment due to their low immunogenicity, toxicity, invasiveness, and cost-effectiveness.¹⁴ Importantly, hucMSC-EVs can promote functional recovery in mice with spinal cord injury by alleviating inflammatory responses.⁵⁷ hucMSC-EVs carrying miR-29a evidently improve ovarian function by targeting HBP1 and activating the Wnt/β-catenin pathway.⁵⁸ In the above studies, the use of hucMSC-EVs shows no toxicities or off-target effects in mice. Mounting studies have revealed the alleviation roles of hucMSCs transplantation in POF animals, as supported by the recovery in ovarian function and hormone levels, improvement in sexual cycle disorder, and an increase in ovarian volume, antral follicle number, and ovarian angiogenesis.^59,6. The above findings elucidated that hucMSC-EVs certainly have a definitive alleviating effect on POF by normalizing estrous cycles and hormone levels.

Figure 2.

hucMSC-EVs ameliorated POF in mice. POF mouse models were induced using D-gal and then treated with hucMSC-EVs, with GW4869 as the control. A) Vaginal cytology assessed estrous cycles in pro-estrus (Pr), estrus (E), metestrus (M), and diestrus (D) over the course of 12 consecutive days. B) ELISA determined the serum levels of FSH, LH, AMH, E2, and P; n=6. Analysis of measurement data was processed using one-way ANOVA, followed by Tukey's test; **p<0.01.

As critical somatic components of the ovary, granulosa cells are necessitated for follicle development by supporting oocyte development and producing disparate growth factors and sex steroids.⁶¹ In mechanism, granulosa cell apoptosis is considered a vital contributor to POF; conversely, inhibition of apoptosis can mitigate POF in animal models.⁶² Owing to the imperative property of ovarian granulosa cells in POF, we conducted in vitro experiments by using the purchased ovarian granulosa cell line KGN. Our results evinced that D-gal-treated KGN cells exhibited decreased proliferation and elevated apoptosis. It is worth noting that hucMSC-derived exosomes can potentiate ovarian granulosa cell proliferation and impede apoptosis.^44,63 In agreement with the preceding research, our findings ascertained that hucMSC-EVs ameliorated the D-gal-induced injury on KGN cells.

Figure 3.

hucMSC-EVs facilitated proliferation and prevented apoptosis of ovarian granulosa cells. The purchased ovarian granulosa cell line KGN was treated with D-gal to induce senescence. A) Immunofluorescence detected the entry of PKH67-labeled hucMSC-EVs into KGN cells. B) CCK-8 assay assessed cell proliferation. C) Flow cytometry assessed cell apoptosis. Cell experimentation was independently repeated thrice. Measurement data were exhibited as mean ±SD. Data analysis was conducted using one-way ANOVA, followed by Tukey's test; **p<0.01.

Intrinsically, E2, a hormone with high biological activity in women, not only shows a considerable role in regulating gene transcription but also activates the PI3K pathway together with estrogen receptors on the cell membrane.⁶⁴ Notably, POI patients have successful pregnancies after grafting the small ovarian fragments that are exposed to PI3K/Akt activators, primarily due to the critical property of the PI3K/Akt/mTOR cascade in follicular recruitment and growth.¹⁵ Moreover, several therapies for POF, such as resveratrol and acupuncture decrease granulosa cell apoptosis in POF rats through the restoration of the PI3K/Akt pathway.^64,65 Preceding research has given rise to the necessity of exploring the primary roles of the PI3K/Akt pathway in KGN cell growth. We noted that p-PI3K/PI3K and p- Akt/Akt were downregulated in D-gal-treated KGN cells but upregulated after the addition of hucMSC-EVs. Consistently, hucMSC-derived microvesicles can upregulate the AKT and p- AKT levels in the ovaries of POI mice.⁶ In addition, a PI3K pathway inhibitor, LY294002 impeded the effects of hucMSCEVs on enhancing KGN cell proliferation and weakening apoptosis. In response to the PI3K/Akt pathway stimulators, mTORC1 in granulosa cells is activated, and subsequently, granulosa cells start to cuboidalize, proliferate, and exclude SMADs proteins.⁶⁷ Compelling evidence suggests a similar mechanism of embryonic stem cell-derived EVs in affecting granulosa cells via promotion in the PI3K/Akt pathway.³⁶ Collectively, hucMSC-EVs potentiated proliferation and restrained apoptosis in KGN cells by activating the PI3K/Akt pathway.

In conclusion, the present study highlighted that hucMSCEVs shortened the estrous cycles of POF mice, facilitated KGN cell proliferation, and repressed apoptosis via activation of the PI3K/Akt pathway, thereby ameliorating POF. On the downside, we only investigated the regulation of hucMSC-EVs on serum sexual hormones in POF mice and ovarian granulosa cells, lacking investigation of animal tissues. The utilization of artificial ovary as a promising alternative to restore fertility has been investigated by many researchers around the world.^68,69 As a human transplantable artificial ovary, it should allow follicular survival and proliferation, facilitate the formation of blood vessels and stroma in vivo, and be safe for the human body.⁷⁰ Future experiments shall be designed to deeply explore the effects of hucMSCEVs on POF using animal tissues or pathological tissues from clinical patients and their precise regulatory mechanisms in the PI3K/Akt cascade. Additionally, more in vivo experiments should be performed to find appropriate transplantable artificial ovarian scaffolds to provide more information for clinical application.

Figure 4.

hucMSC-EVs promoted functional recovery of ovarian granulosa cells through the PI3K/Akt pathway. The PI3K/Akt pathway was blocked using LY294002, a PI3K pathway inhibitor, with DMSO as the control. A) Western blotting determined the protein levels of p-PI3K/PI3K and p-Akt/Akt. B) CCK-8 assay evaluated cell proliferation. C) Flow cytometry evaluated cell apoptosis. Cell experimentation was independently conducted thrice. Measurement data were shown as mean ±SD. One-way ANOVA was employed for data analysis in panel A, followed by Tukey's test. An independent sample t-test was used for data analysis in panels B/C; **p<0.01.

Funding Statement

Funding: this study was supported by a grant from the application of PTEN gene overexpression in the experimental study of improving chemotherapy-induced premature ovarian failure (no. JYTJT20221543).