Exosome-Derived lncRNA LIPE-AS1 for Enhancing Oocytes Maturation and Ameliorating Diminished Ovarian Reserve through Targeting miR-330-5p/HDAC3 Axis

doi:10.21203/rs.3.rs-5159180/v1

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Exosome-Derived lncRNA LIPE-AS1 for Enhancing Oocytes Maturation and Ameliorating Diminished Ovarian Reserve through Targeting miR-330-5p/HDAC3 Axis

https://doi.org/10.21203/rs.3.rs-5159180/v1

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Diminished ovarian reserve (DOR) is a multi-factor gynecological disease that has become a major global health problem. Currently, there is no effective prevention and therapy for DOR. Exosome-drived long non-coding RNAs (lncRNA) in follicular fluid (FF) plays a vital role in development of follicles. Exosome-drived lncRNA LIPE-AS1, which we screened from FF of patients with DOR, regulates histone deacetylase 3 (HDAC3) expression by competitively inhibiting miR-330-5p. Exosomes as nanosized membrane vesicles, could targeted deliver therapeutic agents by modification with target ligands. In this study, we utilize the engineering tochnology to conbime exosome and lncRNA for ovary-targeting therapy of DOR. Firstly, we elucidated the mechanism of lncRNA LIPE-AS1 in occurrence and development of DOR. Secondly, we biologically prepared the exosomes with LIPE-AS1 high expression using 293T cells (Exo-LIPE-AS1). Co-culture of Exo-LIPE-AS1 with oocytes of DOR models promotes oocyte development and improve oocyte quality in vitro. Last, we constructed the FSHβ-modified and LIPE-AS1 loaded exosomes (Exo_FSHβ-LIPE-AS1). The engineered exosomes Exo_FSHβ-LIPE-AS1 could deliver more efficiently to ovary in vitro. In this way, Exo_FSHβ-LIPE-AS1 facilitate the fertility of DOR models. Our research elucidates that exosomes as targeted lncRNA LIPE-AS1 delivery vehicles have potentially preventive and therapeutic effects for DOR.

Health sciences/Diseases/Reproductive disorders/Infertility

Health sciences/Pathogenesis/Clinical genetics/Gene therapy/Targeted gene repair

DOR seriously affects the quality of life of contemporary women, and has become a research hotpots and difficulty in the field of reproductive endocrinology. DOR is diagnosed in women of childbearing age that respond to ovarian stimulation or exhibited lower fertility than their peers, which is mainly manifested as a decrease in the number of follicles in the ovary and a decrease in the quality of oocytes[1]. According to the Bologna criteria laid down by the European Society of Human Reproduction and Embryology, advanced age, abnormal ovarian reserve tests such as antral follicle count (AFC) and anti-Mullerian hormone (AMH), as well as prior suboptimal response to stimulation are the main diagnostic criteria for DOR[2]. DOR is one of the main causes of female infertility[3]. The increasing incidence of infertility has brought tremendous pressure to clinical assisted reproduction[4]. However, in patients with DOR undergoing vitro fertilization - embryo transfer (IVF-ET), decreased ovarian responsiveness, high cycle cancellation rate, decreased number of oocytes obtained, decreased egg quality, and poor embryo development potential were often observed. Low clinical pregnancy rate and low live birth rate are the major problems plaguing clinicians[5, 6].It is urgent to explore effective and safe treatments for DOR.

Follicular fluid (FF), accumulated inside the antral follicle, represents an additional means of communication between oocyte and somatic cells. FF consists of a complex mixture of nucleic acids, proteins, metabolites, and ions, which are secreted by the oocyte, granulose the theca cells, combined with plasma components that cross the blood–follicular barrier via thecal capillaries [7, 8]. FF provides an important microenvironment for oocyte maturation. There are important regulatory factors in follicular fluid that induce cell maturation and provide nutrients for oocytes through intercellular communication[9].

Among the different mechanisms of autocrine and paracrine communication inside the ovarian follicle, an alternative mechanism has recently come to light. Exosomes can transport molecules between body cells / tissues without modifying their contents. The biological roles of exosomes are exerted by either direct communications with surface receptors or by the transportation of their cargo to recipient cells[10, 11]. By this mechanism, the oocyte and somatic follicular cells could interchange signals, as well as, nutrients.

Among different biological signaling molecules, non-coding RNAs (ncRNAs) have the best ability to characterize, probably because they have high stability and are relatively easy to extract compared to proteins. Long noncoding RNAs (lncRNAs) are defined as a subset of transcripts with > 200 nucleotides that are not templates for protein synthesis[12]. lncRNAs are involved in the regulation of gene expression at the epigenetic, transcriptional, and post-transcriptional levels[13, 14]. Existing studies have confirmed the vital functions of lncRNAs in a broad spectrum of physiological and pathological phenotypes. Nowadays, the importance of lncRNAs in occurrence and progression of reproductive related diseases has been widely acknowledged. Many lncRNAs are aberrantly expressed and perform crucial regulatory actions in primary ovarian insufficiency[15], proving their potential as novel therapeutic targets. the competing endogenous RNA (ceRNA) network plays an important role in human fertility[16]. In theory, lncRNAs carry one or more miRNA response elements and can sponge certain miRNAs, thereby upregulating the expression of the miRNA targets.

Exosomes are 50–150 nm size and are secrted by almost all cells. Due to their good safety profile and long circulating half-life, exosomes have attracted widespread interest because the can serve as transport systens to deliver RNAs or chemical drugs for various therapies[17]. In the field of precison nanomedicine, engineered exosomes are promising and effcient drug delivery systems, and their application in clinical trial is credible[18]. In additon, the surface of the exosome can be modified with the desired ligand, such as aptamers or antibodied, to guide it to the site of interest. Research as a targeted drug satisfies that it is expressed only in the reproductive system, followed by minimizing side effects. FSH specifically binds to follicle-stimulationg hormone receptoers (FSHR), which are present on the plasma membrane of ovarian GCs or Sertoli cells in of testes. Current studied show that 1–15, 33–53, 51–65 and 81–95 peptide fragments of FSHβ chain are incolved in receptor binding, of which 33–53 and 81–95 peptides have better recognition and binging ability[19, 20]. The FSH polypeptide and paclitaxel drug delivery nanosystem targeting FSHR were constructed and proved in vivo[21]. We used DSPE-PEG-Mal coupling of FHS-β polypeptides for surface modification of exosomes to achieve exosom-specific delivery of lncRNA to ovarian tissue.

In summary, our study was aimed to screen lncRNA differentially expressed in exosomes in follicular microenvironment of patients with (DOR) and to predic tdownstream target genes as targets for early diagnosis and treatment of DOR. Next, we verified the molecular mechanism of lncRNA competitively inhibiting miR-330-5p toregulate HDAC3 expression in mouse oocytes. Then, he exosomes with over expression of lncRNA LIPE-AS1 was bio-constructed and co-cultured with immature oocytes of mice with to explore the mechanism of Exo-OE-LIPE-AS1 on oocyte maturation in vitro. Finally, we were to explore the effect and mechanism of lncRNA LIPE-AS1-loaded and ovarian-targeted engineered exosomes on ovarian function in mice with DOR.

Patients

According to the study design, female patients receiving IVF treatment at the Reproductive Medicine Center of Ningxia Medical University General Hospital from January 2021 to September 2022 were collected. The diagnostic criteria for DOR patients included in the study were as follows: (1) Age ≤ 35; (2) The number of oocyted obtained is less than 5; (3) FSH > 10UI/L; (4) AMH < 1.1ng/mL. The inclusion criteria of control group: (1) age ≤ 30; (2) The number of eggs obtained is 9–15; (3) FSH < 10UI/L; (4) 2ng/mL < AMH < 6.8ng/mL. A total of 278 cases of funicular fluid in female patients during oocyte pick-up (OPU) were collected, and clinical data such as hormone values of the patients were obtained, including 129 cases of DOR and 149 cases of control group. 3 cases of funicular fluid in each group were used for genome sequencing, 10 cases in each group for sequencing results verification, and the remaining patients were used for correlation analysis.

This study was approved by the Ethics Committee of General Hospital of Ningxia Medical University (Ethics review No.KYLL-2021-235).

Exosome isolation and characterization

The follicle fluid, plasma and culture medium were collected and centrifuged at 3000 g for 15 min to remove cells and cellular debris. Exosomes were isolated using the Exoquick exosome precipitation solution (System Biosciences). The morphology of exosomes was investigated via transmission electron microscopy (TEM; HITACHI, Japen), and the number and size distribution of exosomes were analyzed by nanoparticle tracking anal ysis (NTA) via the NANOSIGHT NS300 system (Malvern, UK). Western blot was utilized to evaluate the expression of exosomal markers

Animals

This study was conducted in strict accordance with the ethical standards set forth by the Animal Ethics Committee of Ningxia Medical University and all processes were carried out according to the Animal Research Guidelines.

6 weeks C57BL/6J female mice were obtained from the laboratory animal center of Ningxia Medical University.Mice were housed in a specific pathogen-free (SPF) conditions at 24 ± 1 ℃, 40–70% humidity and with a 12/12h light/dark cycle, fed and watered ad libitum. Mice were randomly divided into control and DOR groups. The DOR group received intraperitoneal injections of CDDP (2.5 mg/kg/d in saline) (Sigma-Aldrich, Shanghai, China) for 7 consecutive days, and the control group were injected with saline.

Oocyte collection and in vitro maturation

To collect MII oocytes, female mice were injected with 5 IU pregnant mare serum gonadotropin (PMSG) (Ningbo Second Hormone Company, 110254564), and 48 h later with 5 IU human chorionic gonadotropin (hCG) (Ningbo Second Hormone Company, 110251282). After 13.5 h, cumulus-oocyte complexes (COCs) were obtained from the oviducts and MII oocytes were collected by removing the cumulus cells using hyaluronidase digestion (Nanjing Aibei Biotechnology Co., Ltd., M2215).

To collect GV oocytes, female mice female mice were injected intraperitoneally with 5 IU PMSG. After 48 h, ovarian tissues were obtained, COCs were isolated from ovaries in M2 (Sigma-Aldrich, M7167) medium by using a 4.5-gauge needle, and the cumulus cells were cleared away from COCs by repeated mouth-controlled pipetting. The GV oocytes were then cultured with M16 medium (Sigma-Aldrich, M7292) under mineral oil at 37℃, 5% CO2.

In vitro fertilization and embryo culture

Caudae epididymides from 12-week-old male mice were lanced in a dish of HTF medium under mineral oil to release sperm, followed by being capacitated for 1 h (37 ℃, 5% CO2), and added to ovulated oocytes at a concentration of 4 3 105/mL sperm in 100 mL HTF for 5 h at 37℃, 5% CO2. The presence of two pronuclei was scored as successful fertilization. The embryos were cultured in a 96-well culture plate containing 150 mL KSOM under 50 mL mineral oil at 37℃ in a 5% CO2 atmosphere.

Serum hormone levels determination

ELISA was conducted according to the instruction of the mouse AMH, FSH, LH, E2 ELISA Kit (JL20476-96T, JL10239-96T, JL10432-96T, JL11790-96T, Jianglai Bio., China). 50µL of standards with different concentrations were added to each standard well. Serum 10µL and 40µL diluent were added to the sample in the air, and sample diluent 50µL was added to the blank hole. Horseradish peroxidase (HRP) labeled detection antibody was added to 100µL in each well, incubated at 37℃ for 60min, and washed the plate 5 times. Add 50µL of substrate A and B to each well and incubate at 37℃ for 15min without light. Add stopper solution 50µL to each well, measure OD value of each hole at 450nm wavelength within 15min, and draw standard curve.

Estrus cycle examination

The mice underwent daily vaginal exfoliated cell smears from 17:30 to 18:00. 20 µl of saline was gently placed on the vaginal opening and rinsed 2–3 times. The rinsing solution was then coated on slides and observed under a light microscope. Mice estrous cycle was determined by the morphology and type of exfoliated cells for 14 days of observation. Proestrus (P) is characterized by predominantly nucleated epithelial cells, while estrus (E) is characterized by predominantly keratinized and non-nucleated epithelial cells. Metestrus (M) is characterized by a mixture of nucleated epithelial cells, keratinized and non-nucleated epithelial cells, and neutrophils. Diestrus (D) is characterized by a large number of neutrophils.

Histological analysis of ovaries

Ovaries used for histological analysis were collected from each group of mice and fixed in 4%paraformaldehyde (pH 7.5) overnight at 4℃, dehydrated, and embedded in paraffin. Paraffin-embedded ovaries were sectioned at a thickness of 8 mm for hematoxylin and eosin (H&E) staining. Both ovaries from three mice of each group were used for the analysis.

Immunofluorescence of ovaries

The prepared paraffin sections of ovaries were placed in dewaxing solution for 10–15 min and anhydrous ethanol for 15 min. The washed ovarian sections were incubated with EDTA antigen retrieval buffer and repaired in a microwave oven. A histochemical pen was used to draw a circle around the tissue of the slices, and BSA was dropped and incubated for 30 min, following incubating primary antibodies overnight and secondary antibodies for 50 min. DAPI dye solution was incubated at 37°C in darkness for 10 min, and autofluorescence quench was treated for 5 min. The tablets were sealed with anti-fluorescence quench sealing agent. The dewaxed sections were incubated with hematoxylin for 5 min and eosin dye solution for 3 min, then dehydrated and sealed. Finally, the sections were photographed under a fluorescence microscope.

Western blot analysis

A total of around 100 oocytes and ovaries were collected from each group. Each protein sample was extracted using a cell lysis solution containing 1% PMSF (Cell Signaling Technologies) following the manufacturer's instructions. The protein samples underwent boiling at 100°C for 5 min and subsequently separated on a 10% SDS-PAGE gel. Then, proteins were transferred to the polyvinylidene fluoride membrane (Millipore, USA) through a transmembrane transfer. Membranes were immersed in 5% skimmed milk for 2 h at room temperature, washed briefly with TBST (TBS containing 0.1% Tween 20), and incubated with primary antibodies overnight at 4℃. Rinse the membranes 3 times with TBST for 10 min, then incubate them with secondary antibody for 1 h at room temperature and rinse them 3 times with TBST for 15 min each. Finally, the signals were detected using an enhanced chemiluminescence (ECL) kit (KeyGEN BioTECH, KGC4602-200) and quantitatively analyzed in grayscale with Image J.

RNA isolation, reverse transcription, and RT–qPCR

Total RNA was extracted from oocytes and ovaries with TRIzol reagent (Takara, Japan) according to the manufacturer’s protocol, and an equal amount of RNA from each sample was extracted with a reverse-transcription kit (R233,Vazyme, China). Real-time PCR was performed using SYBR® Green gene expression assays (R711, Vazyme, China). GAPDH and U6 were respectively used as the controls for lncRNA LIPE-AS1 and miR-330-5p, and data were collected based on the comparative cycle threshold (CT) (2^−ΔΔCT) method. The primers are listed in Supplementary Table 1.

Transmission electron microscopy (TEM)

Ovary tissues were obtained from 14-week-old mice and fixed in 2.5% glutaraldehyde at 4°C overnight. Subsequently, the tissues were rinsed three times for 10 min each in PBS. Then the ovary was transferred into 1% osmic acid. After 2 h, it was washed three times with PBS for 10 min each time. The ovaries were fixed with 2% uranyl acetate for 30 min and dehydrated in ethanol gradients (50%, 70%, 90%, and 100%) for 10 min each. Transferred to a solution of 90% acetone and 90% ethanol (1:1) for 20 min, then the samples were transferred to 90% acetone at 4°C for 20 min, followed by two transfers to 100% acetone for 15 min each. Finally, they were embedded in epoxy resin. Semi-thin sections after staining with 1% methylene blue in 0.5% borax were examined using an Olympus BX60 light microscope. And ultrathin sections (50 nm), stained by lead citrate, were observed using a HITACHI H-7650 transmission electron microscope (HITACHI, Japan)

Exosome weight suspension 10µL was taken and added to the sample carrying copper network. After precipitation at room temperature for 2-3min, the floating liquid was absorbed on the edge of the copper mesh with filter paper, and then negatively stained with equal volume 3% phosphotungstic acid solution for 4 min, and then absorbed with filter paper. Dry at room temperature for 3 minutes

Immunofluorescence and confocal microscopy

Oocytes were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 30 min and permeablilzed in 0.5% Triton-X-100 for 20 min at room temperature. Then, oocytes were block with 1% BSA-supplemented PBS for 1h and incubated with anti-α-tubulin-FITC antibody (Sigma-Aldrich, F2168), anti-centromere antibody (Antibodies Incorporated, ABI-15-234), anti-ovastacin antibody (Bio-Techne, AF8549) at 4℃ overnight. After washing in PBST, oocytes were incubate with fluorescently labeled secondary antibodies (according to the corresponding primary resistance).Then oocytes were counter-stained with Hoechst for 10 min. Finally, oocytes were mounted on glass slides and observed under a laser scanning confocal microscope (Nikon A1).

For active mitochondrion staining, oocytes were cultured in M16 medium containing 100 nM cell per-meant MitoTracker Red CMXRos (ThermoFisher, M7512) for 30 min at 37℃ in a dark environment and 5% CO2 in air. After washing three times with fresh M2 medium for 20 min each, oocytes were observed under laser scanning con focal microscope.

Mitochondrial membrane potential was evaluated using JC-1 Apoptosis Detection Kit ( KGA1904). oocytes were cultured in M16 medium with JC-1 operating fluid for 30 min at 37 C, followed by washing with buffer for 10 min. Samples were immediately imaged in a glassbottom dish under the laser scanning confocal microscope. JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (530 nm) to red (590 nm). Thus, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.

ROS levels were detected in oocytes using the DCFH-DA (2,7-Dichlorofluorescin Diacetate) probe, following the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, E004-1-1). Briefly, after the oocytes were incubated in DCFH-DA working solution at 37°C for 20 min and washed twice with (phosphate-buffered saline) PBS, the ROS fluorescence signals in oocytes were collected and observed under the laser scanning confocal microscope.

For Annexin-V staining, oocytes or granulosa cells were stained with the Annexin-V Staining Kit (Beyotime, C1062) according to the manufacturer’s instruction. After washing twice in PBS, the viable oocytes were stained for 30 min in the dark with 90 mL of binding buffer containing10 mL of Annexin-V-FITC. Then oocytes were washed three times in DPBS containing 0.1% BSA and placed on glass slides and observed under the laser scanning confocal microscope.

Evaluation of total ATP content

Total ATP content in a pool of 10–30 oocytes was determined using the Bioluminescent Somatic Cell Assay Kit, following the procedure described by Combelles and Albertini (2003) and the manufacturer’s instruction. A 5-point standard curve (0, 0.1, 0.5, 1.0, 10, and 50 pmol of ATP) was generated in each assay and the ATP content was calculated by using the formula derived from the linear regression of the standard curve.

Construction of ovario-targeted exosomes

FSHβ81–95 (DSPE-PEG2000-QCHCGKCDSDSTDCT) was synthesized by Qiang Yao Biotechnology Co., LTD. FSHβ was modified to the exosome surface. 10µL PBS containing Exosome (1012 particles /mL) and 90µL DSPE-PEGMal-FSHβ solution (10µM) were added to 100µL PBS and incubated overnight at 4℃. In order to remove the free DSPE-PEG-Mal-FSHβ, the mixture was centrifuged for 70min at 4°C with a centrifugal force of 120,000g in a ultra-high speed centrifuge, and the precipitation was suspended, thus the ovarian targeted modified exosomes were obtained.

Loading of exosome LIPE-AS1

10µL PBS (1012 particles /mL) containing exosomes and 100µg RNA fragments were mixed in 400µL electroporation buffer (1.15mM potassium phosphate pH 7.2, 25mM potassium chloride, 21% Opti-MEM reduced serum medium). The mixture was placed in a 4mm shock cup, cooled on ice for 6min, and then electroperforated at 0.35s, 20 pulses, and 0.7kV by an electroperforator. After completion, the exosomes were placed at 37°C for 30 min to restore the complete membrane structure of the exosomes. In order to remove the free RNA fragments, the resulting products were subjected to 1.2×105g ultra-high speed centrifugation at 4°C for 90min.

Statistical analysis

Statistical analysis was performed with GraphPad Prism software (GraphPad Software, San Diego, CA). The results are given as the means and standard deviations (SDs). Each experiment included at least three independent samples andwas repeated at least three times. Student’s two-sided t-test was used to calculate the difference between two groups, while difference among multiple groups was calculated by One-way analysis of variance (ANOVA). P values of < 0.05 were considered to indicate statistical significance. The sample sizes (“n”), the statistical tests and the P values were described in each figure legend.

Expression and roles of lncRNA LIPE-AS1 in FF-drived exosomes

Exosomes in follicle fluid were isolated and characterized. TEM and NTA show that the sizes of exosomes from cases were consistent with exosomes frome healthy controls (50–100 nm, Fig. 1A, B). Western blot revealed the presence of the exosome markers, TSG101, CD63 and CD81 (Fig. 1C). The characteristics of 5 candidate lncRNAs are summarized in by genomics sequencing of exosomes in FF (Fig. S1). The AK126698 was delegated in DOR group, while LIPE-AS1, AC010761.9, MAX1-1, DLX6-AS1 were decreased in DOR group compared with control group (Fig. S2). The sequencing results were verified by qRT-PCR, the expression of LIPE-AS1 in exosomes from DOR was significantly lower than that from healthy controls (P < 0.05, Fig. 1D). Correlation analysis (Fig. 1E-G) showed that LIPE-AS1 in FF was positively correlated with AMH level (r = 0.283, P = 0.004). There was no significant correlation between LIPE-AS1 in FF and FSH level (r=-0.107, P = 0.289), and there was a significant positive correlation between LIPE-AS1 in FF and basal follicle (AFC) (r = 0.277, P = 0.005).

LncRNA LIPE-AS1 functions as an endogenous miR-330-5p sponge to facilitate expression of HDAC3

We analyzed the subcellular localization of lncRNA, ant the results were shown in Fig. 2A, lncRNA LIPE-AS1 was mainly located in the cytoplasm of oocytes. When lncRNAs are mainly located in the cytoplasm, they competitively bind specific mirnas through a "molecular sponge" mechanism, thereby relieving the inhibition of miRNAs on their downstream target genes. To predict the potential target miRNAs of lncRNA LIPE-AS1, miR-330-5p was selected from the overlap between the four databases (miRanda, LncBook, LncRBase and starBase). A bioinformatic analysis elucidated that lncRNA LIPE-AS1 shares miRNA response elements with miR-330-5p, and there were complementary sequences of binding sites on both miR-330-5p and HDAC3 mRNA sequences (Fig. 2B). Moreover, the dual-luciferase reporter gene assay verified the sponging role of LIPE-AS1 on miR-330-5p (Fig. 2C). HDAC3 was also confirmed as the target genes of miR-330-5p through dual-luciferase reporter gene assay (Fig. 2C).

Verification result displayed that compared with healthy controls, the expression of LIPE-AS1 in FF was significantly lower in DOR patients (P < 0.01), miR-330-5p was higher (P < 0.01) and HDAC3 was accordingly lower (P < 0.01, Fig. 2D-F). Based on this, we further analyzed the correlation of lncRNA LIPE-AS1 in FF and serum with miR-330-5p, HDAC3 and the results were shown in Fig. S3.

Thus, we sought to investigate whether LIPE-AS1 could regulate HDAC3 levels by competitively binding to miR-330-5p. Firstly, the differential expression of lncRNA LIPE-AS1/miR-330-5p/HDAC3 in MII oocytes of mice in the control group and DOR model mice was verified. As shown in Fig. S4, compared with the control group, the expression of LIPE-AS1 in mice oocytes with DOR was significantly decreased (P < 0.01), the expression of miR-330-5p was significantly increased (P < 0.01), and the expression of HDAC3 protein was decreased (P < 0.001). It was consistent with the expression trend in FF of patients with DOR in clinical cases. Next, we microinjected LIPE-AS1 expressing plasmid vector or si-LIPE-AS1 vector to GV oocytes of mice. And then the oocytes were blocked and cultured in M2 containg IBMX for 12h, and the transplanted into M2 for further culture for next 12h. The results revealed that overexpression of LIPE-AS1 could decrease miR-330-5p expression levels (P < 0.01), accordingly, The high expression of miR-330-5p was followed by the silencing of LIPE-AS1(P < 0.001, Fig. 2H). Last, we microinjected miR-330-5p mimics/inhibitors into GV oocytes of mice, and the expression of HDAC3 at mRNA and protein levels is negatively regulated by miR-330-5p (P < 0.01, Fig. 2I-K)

Si-LncRNA LIPE-AS1 results in impaired oocyte maturation and mitochondrial dysfunction in vitro

In the current study, we attempted to detect the biological role of LIPE-AS1 in vitro, because we found that the downregulated expressiong of LIPE-AS1 in exosomes from follicle fuild. Si-LIPE-AS1 or si-NC was microjected into GV oocytes. Oocytes were cultured in M2 containing IBMX for 12h, and then cultureed in M2 for another 12h. Firstly, the PB1 exclusion rate of oocytes was significantly lower after si-LIPE-AS1 microjection (P < 0.001). Then, MII oocytes were fertilized in vitro. Similarly, the fertilization rate of oocytes with si-LIPE-AS1 injected was significantly lower (P < 0.001). We further monitored the subsequent early embryonic development of fertilized oocytes, revealing the microjection of si-LIPE-AS1 markedly decreased the 4-cell rate (P < 0.01, Fig. 3A-B).

Furthermore, we examined mitochondrial function and the levels of oxidative stress makers, including ROS, MitoTracker and JC-1 of oocyets in each group. We found that ATP levels was markedly decreased after si-LIPE-AS1 injection (P < 0.001, Fig. 3C), and mitochondrial distribution, with mitochondrial depletion (P < 0.01, Fig. 3D-F) were found in oocytes microinjected with si-LIPE-AS1. Accordingly, JC-1 staining showed that mitochondrial membrane potential was significantly decreased (P < 0.001, Fig. 3G,H). and ROS level was also markedly increased in si-LIPE-AS1 group (P < 0.001, Fig. 3I,J).

Isolation and identification of exosomes rich in lncRNA LIPE-AS1

A scheme of collection and isolation of exosomes rich in lncRNA LIPE-AS1 (Exo-LIPE-AS1) was showen in Fig. 4A. We firstly transfected 293T cells with pCDNA-LIPE-AS1. High expressed LIPE-AS1 in the transfected 293T cells was secreted via packing into exosomes. Exo-LIPE-AS1 were isolated and purified from culture medium. LncRNA LIPE-AS1 expression level in the groups pCDNA-LIPE-AS1 vectors could increase more than 20 times when compared with that of the NC vector (Fig. 4B). TEM and NTA were performed again to characterize the morphology and diameter (Fig. 4C,D). Taken together, the above results verified that the type of vesicles in this study was the exosomes. PCR results showed that the expression of LIPE-AS1 in exosomes increased significantly with the transfection of pCDNA-LIPE-AS1 in 293T cells, indicating that Exo-LIPE-AS1 was successfully constructed (Fig. 4E).

Co-culture of Exo-LIPE-AS1 improves the fertilization ability and early embryo development of oocytes of DOR mice

GV oocytes isolated from control and DOR mice were co-culcured in vitro with Exo-LIPE-AS1 and microinjected in vitro with LIPE-AS1 to determine how Exo-LIPE-AS1 improves fertilization ability and early embryo development. Firstly, quantification of the first polar body (PB1) extrusion rate revealed that meiotic progression. The PB1 extrusion rate decreased in DOR group but recovered in group of microinjetion of LIPE-AS1 and Exo-co-culture of IVM. We than whether the fertilization ability of oocytes of DOR mice with Exo-LIPE-AS1 co-culture could be enhanced. An in vitro fertilization experiment showed oocytes of mice in DOR group had dramatically lower fertilization rate compare with ones of mice in control group. Both microinjetion of LIPE-AS1 and Exo-co-culture of IVM could significantly increased the fertilization raote of mice in DOR group, and the effect of co-culture was better than that of microinjection. We further monitored the subsequent early embryonic development of fertilized oocytes, revealing that Exo-co-culture of IVM markedly promoted the 4-cell embryos rate and blastocyst formation rate of fertilized oocytes from DOR mice, however, LIPE-AS1 microinjection had no effect on early embryonic development.

Co-culture of Exo-LIPE-AS1 restores mitochondrial function of oocytes of DOR mice

To verity the effect of on mitochondrial function in oocytes of DOR mice, we first observed the distribution pattern of mitochondria bt MitoTracker staining. We observed that two features of mitochondrial distribution emerged in oocytes of control mice, including accumulated distribution in the periphery of chromosomes and homogeneous distribution in the cytoplasm. In oocytes of DOR mice (Fig. 6A), however, a great number of mitochondria partially or completely lost their accumulation around chromosomes and presented absent or aggregated distribution in the cytoplasm. Quantitatively, more than 30.15% of oocytes of DOR mice exhibited the mislocalized mitochondria, and microjection with LIPE-AS1 and co-culture of Exo-LIPE-AS1 reduced this to 22.92% and 19.62% respectively (Fig. 6B). besides, the fluorescence intensity of MitoTracker of oocytes was restored by co-cultured with Exo-LIPE-AS1 mostly (Fig. 6C). The abnormal distribution of mitochondria indicates that their function might be compromised with oocur of DOR Given that the most important function of mitochondria is to produce ATP for cell development, we measured the ATP content in oocytes from four groups. Our results showed that ATP levels decline prominently in oocytes of DOR mice conpared with control ones but recovered apparently following Co-culture of Exo-LIPE-AS1 more than microinjection with LIPE-AS1 (Fig. 6F). We then assessed the mitochondrial membrane potential (Δψm), the driving force of mitochondrial ATP synthesis, by JC-1 staining (Fig. 6D). Mitochondria with high membrane potential presents red fluorescence, whereas those with low membrane potential exhibited green fluorescence. The ratio of red to green signal was much weaker in oocytes of DOR mice than in control ones but rescued in co-culture of Exo-LIPE-AS1 (Fig. 6E). It is kown that mitochondrial dysfunction is one of the main causes of ROS production and oxidative stress, and the abnormal accumulation of ROS and the accurrence of oxidative stress are important causes of oocyte quality decline. Therefore, oxidative sensitive fluorescent prove DCFH-DA was used to detect ROS (Fig. 6G). Fluorescence imaging and intensity measurements displayed that much stronger ROS signals appeared in oocytes of DOR mice than control ones. On the contrary, co-culture of Exo-LIPE-AS1 and microinjection of LIPE-AS1 effectively reduced accumulated ROS present in oocytes of DOR mice, and the effect of co-cluture was better (Fig. 6H).

Co-culture of Exo-LIPE-AS1 recovers meiotic maturation of oocytes of DOR mice

Because meiotic arrest was mainly due to defective spindle/chromosome structure, we further examined it in oocytes from four groups. As assessed by immunostaining images, a variety of disorganized spindle apparatuses with misaligned chromosomes were present in oocytes of DOR mice at the meiotic (Fig. 7A). The quantitative date showed that the occurrence of aberrant/chromosome structure was considerably higher in oocytes of DOR mice compared with controls but declined after co-culture or microinjection. However, for PB1 extrusion and percentage of aberrant spindle, and the effect of co-culture was better than that of microinjection (Fig. 7B,C).

Co-culture of Exo-LIPE-AS1 rescues the distribution of CGs, DNA damage and apoptosis of oocytes of DOR mice

Cortical granules (CGs) are oocyte-specific vesicles located under the subcortex to block polyspermy following fertilization. It is noteworthy that the distribution of CGs is usually regarded as one of the most important indicators of oocyte cytoplasmic maturation. We assessed whether Exo-LIPE-AS1 would affect the dynamics of CGs in aged oocytes (Fig. 8A). As judged by Lens culinaris agglutinin (LCA)-FITC staining, CGs distributed evenly in the oocyte subcortical region, except the CG-free domain (CGFD) near chromosomes in young oocytes, but lost this normal localization because of discontinuous and weak signals in oocytes of DOR mice. In line with this, the fluorescence intensity of CG signals in oocytes of DOR mice was significantly reduced compared with control ones. The mis-localization and decrease in the amount of CGs were rescued by Co-culture of Exo-LIPE-AS1(Fig. 8B,C).

Because high levels of ROS usually result in accumulation of DNA damage and apoptosis, we next evaluated the DNA damage by detecting γH2A.X expression in oocytes (Fig. 8F) and apoptosis by Annexin-V staining in oocytes (Fig. 8D) of each group. DOR led to a higher incidence of apoptosis in oocytes which was suppressed by co-culture of Exo-LIPE-AS1 and microinjection of LIPE-AS1 (Fig. 8E). Similarly, the expression of γH2A.X in oocytes of DOR mice was significantly lower than oocytes of control group. Both co-culture of Exo-LIPE-AS1 and microinjection of LIPE-AS1 effectively reduced the expression of γH2A.X in oocytes (Fig. 8G).

Preparation and targeting capability and bio-safety of Exo_FSHβ-LIPE-AS1 in vivo

To enhance the targeting capability of exosomes, DSPE-PEG2000-MAL was coupled with the N-terminal mercaptoyl modified ovarian targeting peptide-FSHβ, and the peptide was inserted into the exosome membrane by utilizing the efficient insertion of palmitoyl molecules into the phospholipid bilayer. The nucleotide sequence of LIPE-AS1 was loaded into exosomes by electroporation technique, and obtained the suitable engineered exosomes named Exo_FSHβ-LIPE-AS1. The morphology of Exo-LIPE-AS1 and Exo_FSHβ-LIPE-AS1 were investigated by TEM (Fig. 9A), that showed round spherical vesicles. The mean diameters of Exo_FSHβ-LIPE-AS1 were 132.5 ± 12.9 nm by NTA (Fig. 9B), which showed no significant difference with that of Exo-LIPE-AS1. Then, we detected the nucleic acid drug release rate of Exo_FSHβ-LIPE-AS1, which was about 23.81 ± 4.37% (Fig. 9C).

We further assessed the ovary-targeting and the accumulation ability in vivo. The Exo_FSHβ-LIPE-AS1 and Exo-LIPE-AS1 labeled with DIR iodide fluorescent dye were intravenously injected into nude mice, and the fluorescence intensity was tested at 1h, 12h, 24h by in vivo imaging system. As shown in Fig. 9E, the fluorescence intensity in the ovary sites of Exo_FSHβ-LIPE-AS1 group was significantly stronger than that of Exo-LIPE-AS1 group. In addition, the fluorescence intensity gradually increased at 1h after injection and was the strongest at 12h. At 24h, the fluorescence intensity decreased, indicating that Exo_FSHβ-LIPE-AS1 began to gradually metabolize in mice at 24h.

Meanwhile, the blood routine and biochemical analysis indicated the negligible effect on the liver and kidney function biomarkers (Alaninetransaminase (ALT), Aspartate transaminase (AST) and Blood urea nitrogen (BUN), Creatinine (CREA)) in the mice with injection of Exo_FSHβ-LIPE-AS1 (Fig. 10A-B). H&E staining demonstrated negligible histological changes on normal organs (heart, liver, spleen, lung and kidney), demonstrating the excellent bio-safety of the engineered exosome (Fig. 10C). Finally, we tested the nucleic acid release characteristics of Exo_FSHβ-LIPE-AS1, as shown in Fig. 10D. At 2h, the nucleic acid release rate reached 12%, and with the passage of time, the drug release rate tended to flatline, reaching 91% at 24h.

Exo_FSHβ-LIPE-AS1 partially ameliorates the DOR phenotype by targeting miR-330-5p/HDAC3

We first evaluated the effect of CPDD on ovarian damage in mice. For follicle count, by day 3, the CPDD-treated group had significantly fewer antral follicles. By day 5, the CPDD-treated group had significantly fewer primordial follicles, secondary follicles and antral follicles, and more atresia follicles. By day 7 the longer exposure to CDDP more seriously damaged the primodial, secondary and antral follciles and produced excessive atresia follicles than the PBS-treated group. According to the time point of the reduction of primordial follicles, we added Tail intravenous injection of Exo_FSHβ-LIPE-AS1 on day 5 of CPDD to act as a preventive therapy for DOR (Fig. 11A).

Intriguingly, compared with the control group, the ovarian tissue of mice with DOR model showed different degrees of atrophy, and the structure of follicles was destroyed (Fig. 11C). Exo_FSHβ-LIPE-AS1 intervention improved the number and morphological defects of follicles in mice with DOR and Exo_FSHβ-LIPE-AS1 intervention restored the ovary weight of DOR mice to normal levels (Fig. 11D-E). In addition, on assessment of estrous cycle, DOR mice exhibited disordered estrous cycle with prolonged diestrus compared with control ones, but Exo_FSHβ-LIPE-AS1 intervention partially improved this disorder and significantly shortened the duratuib of diestrus (Fig. 11B). we evaluated the oocyte morphology of mice in three groups. We found that maternal aging dramatically reduced the number of ovulated oocytes and the rate of matured oocytes but increased the incidence of fragmented oocytes, Conversely, Exo_FSHβ-LIPE-AS1 intervention apparently ameliorated the defects in the number and morphology of oocytes caused by DOR. In addition, we assessed follicle development by ovary sections. (Fig. 11F-H). Funicular development and periodic ovulation are regulated by sex hormones secretion. The serum hormone assay indicated significant decrease of AMH, E₂ and increase of FSH in the DOR group. After Exo_FSHβ-LIPE-AS1 treatment, the serum AMH and E₂ levels increased significantly, nearly reaching to the control group, which indicated the recovery of ovarian function (Fig. 11K-M). Moreover, the Exo_FSHβ-LIPE-AS1 intervention partially improved the CDDP-induced increase of atretic follicles and the decrease of secondary follicles and sinus follicles (Fig. 11I,J).

Finally, we mated each separate group of mice with healthy and fertile male mice for a 7-month fertility experiment. The outcomes demonstrated a significant upturn in both cumulative offspring count and average number of litters for the Exo_FSHβ-LIPE-AS1 group. In conclusion, these findings highlight that Exo_FSHβ-LIPE-AS1 partially ameliorates reproductive function of DOR models (Fig. 11O,P).

The expression of lncRNA LIPE-AS1 in ovarian tissue of DOR model mice was significantly lower than that of the control group. With the increase of LIPE-AS1 expression in vivo supplementation with Exo_FSHβ-LIPE-AS1, the expression of miR-330-5p was decreased and the expression of HDAC3 was increased (Fig S6).

Nowadays, a growing number of studies have demonstrated the regulative roles of lncRNA in the oocyte maturation and ovarian function[22, 23]. DOR is a multi-factor induced disease, the causes of which include genetic, immune, infection, environment, androgenic factors and bad living habits[24, 25]. We identified that lncRNA LIPE-AS1 was mainly in exosomes of FF, and found that LIPE-AS1 was downregulated in DOR patiens. lncRNA LIPE-AS1, located on human chromosome 19, is the antisense gene of LIPE, the gene encoding hormonesensetive lipase (HSL), which is mainly invovled in the decomposition of diglycerol into monoglycerol. Besides, LIPE-AS1 is highly conserved between mice and human, and its expression has common regilatory effects and fuction[26].Additionally, this study showed that LIPE-AS1 could be packaged into exosomes and secrted it into oocyetes of the DOR mice modle, which significantly prompte oocyte maturation in vitro. Exosomal LIPE-AS1 also Improve ovarian reserve in vivo. Thus, exosomal LIPE-AS1 could be a potenial candidate of DOR dectection. In addition, we confirmed that exosomal LIPE-AS1 regulate HDAC3 by sponging miR-330-5p.

IVM technology of oocytes was established in the 1930s[27, 28], and first major advances in the field of IVM, on which IVF technology was built, was made in 1960s[29]. But at present, IVM of human oocytes is only uesed in a few centers around the world. IVM medium has formed a certain formula to provide nutrients for oocyte development potential of mature oocytes in vitro is largely inferior to that of mature oocyets in vivo[30–32]. The main challenge for IVM is that oocyte in vitro exhibit lower developmental potential compared to oocytes in vivo[33]. In all mammals. The follicular origin of the oocytes has an irreplaceable influence on its subsequent developmental potential. Once the oocyted is removed from its follicle too early, its developmental potential is impaired, which was considered an obstacle that had to be overcome for IVM success[34].

The extraction of oocyte from the follicular microenviroment is inevitable during IVF-ET treatment, which is followed by loss of GCs and FF. However, current studied have shown that the effective measures can be taken to mitigate the adverse effects of IVM of oocyt. The first is to mimic the in vivo follicular environment of oocytes as much as possilbe in vitor, including maintaining the structural integrity of COCs and maintaining meiosis stasis[35]. Basing on co-culture with natriutrtic peptides (NPs) and cAMP hydrolytic inhibitors (e.g. IBMX), it can effectively prevent oocyte meiosis recovery and preserve COC structure. The second method is to add growth factors of follicular microenvironment, including epidermal growth factor (EGF), which is upregated during oocyte maturation. Research showed that adding growth factors in IVM medium can obtain oocytes of better quality[36]. Therefore, current IVM studies should more closely follow interactions of GCs-oocytes.

293T cells line is widly used as tool cells. We first constructed a 293T cell line with lncRNA LIPE-AS1 overexpression, and then ectracted exosomes from the culture supernatant. PCR dectected that LIPE-AS1 in exosomes was also highly expressed, whichi could be used for co-incubation of oocytes in vitro. the concentration of follicular fluid derived exosomes in IVM mediun of oocytes was discussed in previous[37]. The exosomes extracted from follicular fluid of comparable volume were added to IVM base medium (exosomes extracted from 1 mL folliclar fluid were added to 1mL IVM mediun, ie 1:1) or diluted 2 times (exosomes extracted from 1 mL folliclar fluid were added to 2 mL IVM mediun, ie 2:1) or concentrated 2 times (1:2) or concentrated 4 times (1:4). Our results showed that the average particle concentration of exosomes in follicular fluif of control group was 32.28 ± 6.45 × 10⁹ particles/mL, and the average paticle concentration of exosomes extracted from 293T cell line was 7.04 ± 3.86 × 10⁹ particles/mL. We designed four exosome concentration gradients: the concentration of exosome particles extracted from the culture supernatant of 293T cell line per 4mL was added into every 1ml IVM mediun (1:1), 1:2 was 2mL, 2:1 was 8mL and 4:1 was 16mL. Considering exosome extraction efficiency and effect of oocyte IVM, 2:1 was selected as the optimal concentration for IVM (Fig S7).

We further discover that Exo-OE-LIPE-AS1 elevated the maturation rate of oocytes of DOR mice modles by promoting nuclear and cytoplasmic maturatiom. Co-culture of Exo-OE-LIPE-AS1 recoved the spingdle/chromosome structure to maintian euploidy and nuclear maturation of oocytes of DOR mice. Moreover, Co-culture of Exo-OE-LIPE-AS1 restored cytoplasmic maturation of oocytes of DOR mice by ensuring normal dynamics of CGs and their component ovastacin. Researches hace revealed that genes related that aging disrupts mitochondrial funciton in a variety of cells[38, 39]. During oogenesis, mitochodrial numbers increase rapidly, while the rate of oxygen consumption is maintained at a low level[40]. patients with POI exhibit increases in both mitochondria mutation and ROS levels[41, 42]. Oxidative stress and mitochondrial dysfunction induce ovarian cell apoptosis to contribute to ovarian aging[43, 44]. indiced by DOR. Reduced Δψm and compromised mitochondrial function predict accumulation of ROS and apoptosis in aged oocytes, which accounts for their high frequency of fragmentation. Our data showed that co-culture of Exo-LIPE-AS1 could reverse mitochondrial dysfunction and efficiently eliminate excessive ROS to suppress DNA damage and apoptosis.

Evidence that exosomes were acting on in the present experiment was the finding that exosomes in FF increased cumulus expansion and cumulus cell proliferation[45–47]. Besides, exosomes derived from plasma and oviductal also increased the proportion of COCs exhibiting cumulus expansion as well as cumulus cell proliferation and maturation rate of oocytes[48, 49].

Exosomes-based therapy ia an emerging and promising strategy, which could circumvent many issues associated with traditional cell therapy, including immune rejection, biocompatibility, high costs, and long therapeutic times.

The physical preparation of engineered exosomes includes the use of ultrasound, electroporation, freeze-thaw and other methods to destroy the intefrity of the cell membrane and make it easier for molecules to enter the exosomes. Electroporation is a well-utilized method for passive packaging. The process forms temporary pores in the exosome membrane by an electric field, through which the drugs diffuse, and then the integrity of the exosomal membrane is restored. Electroporation has been used to encapsulate all kinds of cargoes[50]. miR-155 was loaded into exosomes through electroporation, which significantly increased the level of miRNA-155 in mouse liver[51]. And the subcutaneous tumor model and the orthotopic tumor model in prostate cancerresponded well to the engineered exosomes with siRNA by electroporation[52].The surface molecules anchored on exosomes of different cell and tissue origin are different, which gives the exosomes selectivety for specific receptor cells. The surface modification of exosomes at the site of the diseased tissue, and minimize the toxic side effects while maximizing the therapeutic effect. Here, surface modification of exosomes with FSHβ polypeptide couled with DSPE-PEG-Mal was performed to achieve specific delivery of lncRNA to ovarian. And then we investigated its safety and observed the effective ovarian-targeting capability in vitro.

We evalutated the effect of Exo_FSHβ-LIPE-AS1on ovarian reserve in DOR mice. Overall, to our knowledge, our current work is the first bio engineered exosomes-based, which provides an innovative solution for prevention and treatment of DOR and lays a solid foundation for future clinical applications.

In this study, the differential expression profiles of lncRNA and mRNA in follicular fluid exosomes of patients with DOR were determined by RNA sequencing, and the expression and subcellular localization of lncRNA LIPE-AS1 in oocytes were verified. Bioinformatics is used to predict regulatory networks of ceRNAs that cause DOR. The regulatory and targeted binding relationships of lncRNA LIPE-AS1/miR-330-5p/HDAC3 were verified at the cellular level. To elucidate the effect of lncRNA LIPE-AS1 competitive combination with miR-330-5p regulating HDAC3 expression on the quality and maturation of mice oocytes with DOR. Secondly, exosomes with high expression of lncRNA LIPE-AS1 were constructed and co-cultured with DOR mouse oocytes to explore the role of exosomes IVM of oocytes. Finally, ovarian targeted engineered exosomes loaded with lncRNA LIPE-AS1 were constructed to treat DOR model mice in vivo, and the improvement effect of engineered ovarian targeted exosomes on ovarian reserve function of DOR mice was evaluated. In this study, exosomes in the ovarian microenvironment were used to solve the problem of decreasing oocyte quantity and quality in patients with DOR, and a new application model of exosomes in oocyte IVM and in vivo treatment of DOR was proposed, providing a new idea for clinical diagnosis and treatment of such patients.

This study was approved by the Ethics Committee of General Hospital of Ningxia Medical University (Ethics review No.KYLL-2021-235). All patients were informed of the purpose and methods of the experiment and signed the informed consent.

Acknowledgements

We would like to express heartfelt gratitude to our colleagues in Reproductive Medicine Center, General Hosptial of Ningxia Medical University, Ningxia Medical University and Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education.

Authors’ contributions

Jialing Li and Rong Hu conceived the study. Jialing Li, Miaomiao Tian, Hua Guo, Feimiao Wang, Lu Wang, Jinmei Gao conduct an experiment. Jialing Li and Jie Ma contributed to statistical analyses. Feimiao Wang and Hua Guo participated in the interpretation of the data and critical revision. Jialing Li drafted the manuscript. All authors were involved in revising the article and have approved this final version.

Funding statement

This study was funded by National Natural Science Foundation of China (No.82160293), Key Research and Development Program of Ningxia Hui Autonomous Region (2022BEG0202) and Natural Natural Science Foundation of Ningxia (2022AAC02006).

Data availability

On reasonable request, the corresponding author will provide all datasets used and/or analyzed during the current work.

Consent for publication

Not applicable.

Competing interests

None of the authors have any competing interests to declare.

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Exosome-Derived lncRNA LIPE-AS1 for Enhancing Oocytes Maturation and Ameliorating Diminished Ovarian Reserve through Targeting miR-330-5p/HDAC3 Axis

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Abstract

Figures

Introduction

Materials and methods

Patients

Exosome isolation and characterization

Animals

Oocyte collection and in vitro maturation

In vitro fertilization and embryo culture

Serum hormone levels determination

Estrus cycle examination

Histological analysis of ovaries

Immunofluorescence of ovaries

Western blot analysis

RNA isolation, reverse transcription, and RT–qPCR

Transmission electron microscopy (TEM)

Immunofluorescence and confocal microscopy

Evaluation of total ATP content

Construction of ovario-targeted exosomes

Loading of exosome LIPE-AS1

Statistical analysis

Results

Expression and roles of lncRNA LIPE-AS1 in FF-drived exosomes

LncRNA LIPE-AS1 functions as an endogenous miR-330-5p sponge to facilitate expression of HDAC3

Si-LncRNA LIPE-AS1 results in impaired oocyte maturation and mitochondrial dysfunction in vitro

Isolation and identification of exosomes rich in lncRNA LIPE-AS1

Co-culture of Exo-LIPE-AS1 restores mitochondrial function of oocytes of DOR mice

Co-culture of Exo-LIPE-AS1 recovers meiotic maturation of oocytes of DOR mice

Preparation and targeting capability and bio-safety of ExoFSHβ-LIPE-AS1 in vivo

ExoFSHβ-LIPE-AS1 partially ameliorates the DOR phenotype by targeting miR-330-5p/HDAC3

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

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Preparation and targeting capability and bio-safety of Exo_FSHβ-LIPE-AS1 in vivo

Exo_FSHβ-LIPE-AS1 partially ameliorates the DOR phenotype by targeting miR-330-5p/HDAC3