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Background Although many reports show that various kinds of stem cells have the ability to recover the function of premature ovarian insufficiency (POI), few studies are associated with the mechanism of stem cell treatment of POI. We designed this experimental study to investigate whether human adipose stem cell-derived exosomes (hADSC-Exos) retain the ability to restore ovarian function and how hADSC-Exos work in this process. Methods A POI mouse model was established and human ovarian granule cells (hGCs) collected from individuals with POI were prepared to assess the therapeutic effects and illuminate the mechanism of hADSCs in curing POI. The hematoxylin and eosin assay method was employed to assess the number of follicles. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of sex hormones. The proliferation rate and marker expression levels of hGCs were measured by flow cytometry (fluorescence-activated cell sorting). Real-time PCR and western blot assays were used to determine the mRNA and protein expression levels of SMAD2, SMAD3, and SMAD5. Western blot assays were used to test the protein expression levels of apoptosis genes (Fas, FasL, caspase-3, and caspase-8). Results After the hADSC-Exos were transplanted into the POI mice model, they exerted better therapeutic activity on mouse ovarian function, improving follicle numbers during four stages. ELISA results showed that hADSC-Exos elevated the hormone levels to the normal levels. In addition, after hADSC-Exos were cocultured with POI hGCs, our results showed that hADSC-Exos significantly promoted the proliferation rate and inhibited the apoptosis rate. Furthermore, hADSC-Exos also increased the marker expression of hGCs to the normal level. Besides, mRNA and protein assays demonstrated that hADSC-Exos downregulated the expression of SMAD2, SMAD3, and SMAD5 in vivo and in vitro. Western blot assay demonstrated that hADSC-Exos inhibited expression of the apoptosis genes in POI hGCs, and SMAD knockdown increased the protein expression of apoptosis genes. Conclusions These findings demonstrate for the first time the molecular cascade and related cell biology events involved in the mechanism by which exosomes derived from hADSCs improved ovarian function of POI disease via regulation of the SMAD signaling pathway.

The most effective method for mapping N6-methyladenosine (m 6 A) is m 6 A RNA immunoprecipitation sequencing (MeRIP-seq). The quality of MeRIP-seq relies on various factors, with the anti-m 6 A antibody being a crucial determinant. However, comprehensive research on anti-m 6 A antibody selection and optimal concentrations for different tissues has been limited. In this study, we optimized the concentration of five different anti-m 6 A antibodies across various tissues. Our findings demonstrated that 5 µg of Millipore antibodies (ABE572 and MABE1006) performed well, starting from 15 µg total RNA from the liver, while 1.25 µg of Cell Signaling Technology antibodies (CST) (#56593) was suitable for low-input total RNA. In summary, we provide a significant guideline for anti-m 6 A antibody selection in MeRIP sequencing for different tissues, especially in the context of low-input RNA.

Background Premature ovarian insufficiency (POI) is one of the major causes of infertility. We previously demonstrated that transplantation of menstrual blood-derived stromal cells (MenSCs) effectively improved ovarian function in a murine model of POI. Recent studies indicated that mesenchymal stem cell-derived exosomes were important components in tissue repair. In this study, we investigated the therapeutic effects of MenSCs-derived exosomes (MenSCs-Exos) in a rat model of POI and its mechanism in restoring ovulation. Methods Ovaries of 4.5-day-old Sprague Dawley rats (SD rats) were cultured in vitro to evaluate the effects of MenSCs-Exos exposure on early follicle development. Furthermore, POI in rats was induced by intraperitoneal administration of 4-vinylcyclohexene diepoxide (VCD). Forty-eight POI rats were randomly assigned to four groups, each receiving a different treatment: PBS, MenSCs, MenSCs-Exos, and Exo-free culture supernatant of MenSCs. Estrous cyclicity, ovarian morphology, follicle dynamics, serum hormones, pregnancy outcomes, and molecular changes were investigated. Results Exposure to MenSCs-Exos promoted the proliferation of granulosa cells in primordial and primary follicles in vitro and increased the expression of early follicle markers Deleted In Azoospermia Like (DAZL) and Forkhead Box L2 (FOXL2) while inhibiting follicle apoptosis. In vivo, MenSCs-Exos transplantation effectively promoted follicle development in the rat model of POI and restored the estrous cyclicity and serum sex hormone levels, followed by improving the live birth outcome. In addition, transplantation of MenSCs-Exos regulated the composition of the ovarian extracellular matrix and accelerated the recruitment of dormant follicles in the ovarian cortex and increased proliferation of granulosa cells in these follicles. Conclusion MenSCs-Exos markedly promoted follicle development in vitro and in vivo and restored fertility in POI rats, suggesting a restorative effect on ovarian functions. The therapeutic effect of MenSCs-Exos transplantation was sustainable, consistent with that of MenSCs transplantation. Our results suggested that MenSCs-Exos transplantation may be a promising cell-free bioresource in the treatment of POI.

Background With the development of regenerative medicine and tissue engineering technology, almost all stem cell therapy is efficacious for the treatment of premature ovarian failure (POF) or premature ovarian insufficiency (POI) animal models, whereas little stem cell therapy has been practiced in clinical settings. The underlying molecular mechanism and safety of stem cell treatment in POI are not fully understood. In this study, we explored whether fetal mesenchymal stem cells (fMSCs) from the liver restore ovarian function and whether melatonin membrane receptor 1 (MT1) acts as a regulator for treating POI disease. Methods We designed an in vivo model (chemotherapy-induced ovary damage) and an in vitro model (human ovarian granulosa cells (hGCs)) to understand the efficacy and molecular cues of fMSC treatment of POI. Follicle development was observed by H&E staining. The concentration of sex hormones in serum (E2, AMH, and FSH) and the concentration of oxidative and antioxidative metabolites and the enzymes MDA, SOD, CAT, LDH, GR, and GPx were measured by ELISA. Flow cytometry (FACS) was employed to detect the percentages of ROS and proliferation rates. mRNA and protein expression of antiapoptotic genes (SURVIVIN and BCL2), apoptotic genes (CASPASE-3 and CASPASE-9), and MT1 and its downstream genes (JNK1, PCNA, AMPK) were tested by qPCR and western blotting. MT1 siRNA and related antagonists were used to assess the mechanism. Results fMSC treatment prevented cyclophosphamide (CTX)-induced follicle loss and recovered sex hormone levels. Additionally, fMSCs significantly decreased oxidative damage, increased oxidative protection, improved antiapoptotic effects, and inhibited apoptotic genes in vivo and in vitro. Furthermore, fMSCs also upregulated MT1, JNK1, PCNA, and AMPK at the mRNA and protein levels. With MT1 knockdown or antagonist treatment in normal hGCs, the protein expression of JNK1, PCNA, and AMPK and the percentage of proliferation were impaired. Conclusions fMSCs might play a crucial role in mediating follicular development in the POI mouse model and stimulating the activity of POI hGCs by targeting MT1.

Background Before starting gonadotoxic therapies, cryopreservation of mature sperm has been proposed worldwide as a method for male fertility preservation and for enabling the conception of a healthy baby with assisted reproductive technology (ART); however, these technologies are not feasible for prepubertal boys and men with spermatogenic failure. Transplantation of mesenchymal stem cells has exhibited successful therapeutic benefits in restoring spermatogenesis via gonadal graft angiogenesis, transplanted cell clonogenesis, and disordered somatic compartment recovery. This study aimed to elucidate the fertility protective effects and the underlying mechanisms of human amnion mesenchymal stem cells (hAMSCs) against busulfan-induced testis toxicity. Methods An in vivo busulfan-induced testis toxicity mouse model and an in vitro busulfan-administered mouse Sertoli cell line were employed to evaluate the efficacy and mechanisms of hAMSC transplantation on male fertility preservation. The process of spermatogenesis was evaluated histologically, and the percentage of seminiferous tubules with vacuoles was evaluated by HE staining. Semen parameters were calculated by computer-assisted semen analysis. ELISA was employed to test the testosterone concentration and the levels of oxidative- and antioxidative-associated substances LDH, MDA, GR, SOD, GPx, and CAT. The rates of proliferation (Ki67), apoptosis (Annexin V), and ROS were measured by FACS. The fluorescence intensity of a marker of apoptosis (TUNEL) and a meiosis gene in spermatogenesis (SCP3) were detected by immunofluorescence assay. The expression of mRNA in germ cell-specific (GCS) genes (Dazl, Ddx4, and Miwi) and meiosis genes (Scp3, Cyclin A1, and Stra8) was tested by qPCR. The expression of antiapoptotic proteins (SURVIVIN and BCL2), apoptotic proteins (CASPASE3 and CASPASE9), GCS proteins (Dazl, Ddx4, and Miwi), and meiosis proteins (Scp3, Cyclin A1, and Stra8) was tested by western blotting. Results hAMSC transplantation following disruption by busulfan-induced testis toxicity restored spermatogenesis, elevating testosterone levels and enhancing testicular weight, size, and semen parameters in vivo. In addition, hAMSCs clearly ameliorated cell apoptosis, enhanced cell proliferation, repressed oxidative damage, and augmented oxidative defense in vivo and in vitro. Moreover, hAMSCs distinctly increased the expression of the GCS genes Dazl, Ddx4, and Miwi and the meiosis genes Scp3, Cyclin A1, and Stra8 in vivo. Conclusions hAMSCs might represent a promising tool for the use in regenerative medicine, as these cells can restore spermatogenesis in a busulfan-induced testis toxicity mouse model and facilitate activity in a busulfan-administered mouse Sertoli cell line by resisting apoptosis and oxidative stress.

Background/Aims: Human adipose-derived stem cells (hADSCs) are a potential therapeutic option for clinical applications because of their ability to produce cytokines and their capacity for trilineage differentiation. To date, few researchers have investigated the effects of hADSCs on natural ovarian aging (NOA). Methods: An NOA mouse model and human ovarian granule cells (hGCs) collected from individuals with NOA were prepared to assess the therapeutic effects and illuminate the mechanism of hADSCs in curing NOA. Enzyme-linked immunosorbent assay was used to detect the serum levels of sex hormones and antioxidative enzymes. The proliferation rate and marker expression level of hGCs were measured by flow cytometry (FACS). Cytokines were measured by a protein antibody array methodology. Western blot assays were used to determine the protein expression levels of SIRT1 and FOXO1. Results: Our results showed that hADSCs displayed therapeutic activity against ovarian function in an NOA mouse model, increasing the proliferation rate and marker expression level of hGCs. Furthermore, the yields of hADSC-secreted HGF and bFGF were higher than those of other growth factors. FACS showed that combination treatment with the growth factors HGF and bFGF more strongly promoted proliferation and inhibited apoptosis in hGCs than HGF or bFGF treatment alone. FACS and ELISA revealed that the combination treatment with both growth factors inhibited oxidative stress more forcefully than treatments with only one of these growth factors. In addition, protein assays demonstrated that combination treatment with both growth factors suppressed oxidative stress by up-regulating the expression of SIRT1 and FOXO1. Conclusion: These findings demonstrate for the first time the molecular cascade and related cell biology events involved in the mechanism by which HGF and bFGF derived from hADSCs improved ovarian function during natural aging via reduction of oxidative stress by activating the SIRT1/FOXO1 signaling pathway.

The growth and maturation of oocyte is accompanied by the accumulation of abundant RNAs and posttranscriptional regulation. N6-methyladenosine (m 6 A) is the most prevalent epigenetic modification in mRNA, and precisely regulates the RNA metabolism as well as gene expression in diverse physiological processes. Recent studies showed that m 6 A modification and regulators were essential for the process of ovarian development and its aberrant manifestation could result in ovarian aging. Moreover, the specific deficiency of m 6 A regulators caused oocyte maturation disorder and female infertility with defective meiotic initiation, subsequently the oocyte failed to undergo germinal vesicle breakdown and consequently lost the ability to resume meiosis by disrupting spindle organization as well as chromosome alignment. Accumulating evidence showed that dysregulated m 6 A modification contributed to ovarian diseases including polycystic ovarian syndrome (PCOS), primary ovarian insufficiency (POI), ovarian aging and other ovarian function disorders. However, the complex and subtle mechanism of m 6 A modification involved in female reproduction and fertility is still unknown. In this review, we have summarized the current findings of the RNA m 6 A modification and its regulators in ovarian life cycle and female ovarian diseases. And we also discussed the role and potential clinical application of the RNA m 6 A modification in promoting oocyte maturation and delaying the reproduction aging.

Background The side effects of busulfan on male reproduction are serious, so fertility preservation in children undergoing busulfan treatment is a major worldwide concern. Human placental mesenchymal stem cells (hPMSCs) have advantages such as stable proliferation and lower immunogenicity that make them an ideal material for stimulating tissue repair, especially restoring spermatogenesis. The protective effects of hPMSCs in busulfan-induced Sertoli cells and in busulfan-treated mouse testes have not been determined. Our study aimed to elaborate the protective effect and potential mechanisms of hPMSCs in busulfan-treated testes and Sertoli cells. Methods First, we developed a mouse model of busulfan-induced testicular toxicity in vivo and a mouse Sertoli cell line treated with busulfan in vitro to assess the protective effect and mechanisms of hPMSC treatment on spermatogenesis. Then, the length, width, and weight of the testes were monitored using Vernier calipers. Furthermore, at 1 week and 4 weeks after the transplantation of hPMSCs, histological sections of testes were stained with hematoxylin-eosin, and the seminiferous tubules with fluid-filled cavities were counted. Through ELISA analysis, testosterone levels and MDA, SOD, LDH, and CAT activities, which are associated with ROS, were detected. Markers of ROS, proliferation (Ki67), and apoptosis (Annexin V) were evaluated by FACS. Next, the fluorescence intensity of proliferation markers (BrdU and SCP3), an antioxidant marker (SIRT1), a spermatogenesis marker (PLZF), and autophagy-related genes (P62 and LC3AB) were detected by fluorescence microscopy. The mRNA expression of γ-H2AX, BRCA1, PARP1, PCNA, Ki67, P62, and LC3 was determined by qRT-PCR. Results hPMSCs restored disrupted spermatogenesis, promoted improved semen parameters, and increased testosterone levels, testis size, and autophagy in the testis toxicity mouse model induced by busulfan. hPMSCs suppressed the apoptosis of Sertoli cells and enhanced their rate of proliferation in vitro. Additionally, hPMSCs protected against oxidative stress and decreased oxidative damage in the testis toxicity mouse model induced by busulfan. Furthermore, hPMSCs increased the expression of proliferation genes (PCNA and KI67) and decreased the mRNA levels of apoptotic genes such as γ-H2AX, BRCA1, and PARP1. Conclusions This research showed that hPMSC injection ameliorated busulfan-induced damage in the testis by reducing apoptosis/oxidative stress and promoting autophagy. The present study offers an idea for a new method for clinical treatment of chemotherapy-induced spermatogenesis.

Background Although many reports show that various kinds of stem cells have the ability to recover function in premature ovarian aging, few studies have looked at stem cell treatment of natural ovarian aging (NOA). We designed this experimental study to investigate whether human amniotic mesenchymal stem cells (hAMSCs) retain the ability to restore ovarian function, and how hAMSCs work in this process. Methods To build the NOA mouse model, the mice were fed for 12–14 months normally with young fertile female mice as the normal control group (3–5 months old). Hematoxylin and eosin staining permitted follicle counting and showed the ovarian tissue structure. An enzyme-linked immunosorbent assay was used to detect the serum levels of the sex hormones estradiol (E2), anti-mullerian hormone (AMH), and follicle-stimulating hormone (FSH). The proliferation rate and marker expression level of human ovarian granule cells (hGCs) (ki67, AMH, FSH receptor, FOXL2, and CYP19A1) were measured by flow cytometry (FACS). Cytokines (growth factors) were measured by a protein antibody array methodology. After hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were co-cultured with hGCs, proliferation (ki67) and apoptosis (Annexin V) levels were analyzed by FACS. After HGF and EGF were injected into the ovaries of natural aging mice, the total follicle numbers and hormone levels were tested. Results After the hAMSCs were transplanted into the NOA mouse model, the hAMSCs exerted a therapeutic activity on mouse ovarian function by improving the follicle numbers over four stages. In addition, our results showed that hAMSCs significantly promoted the proliferation rate and marker expression level of ovarian granular cells that were from NOA patients. Meanwhile, we found that the secretion level of EGF and HGF from hAMSCs was higher than other growth factors. A growth factor combination (HGF with EGF) improved the proliferation rate and inhibited the apoptosis rate more powerfully after a co-culture with hGCs, and total follicle numbers and hormone levels were elevated to a normal level after the growth factor combination was injected into the ovaries of the NOA mouse model. Conclusions These findings provide insight into the notion that hAMSCs play an integral role in resistance to NOA. Furthermore, our present study demonstrates that a growth factor combination derived from hAMSCs plays a central role in inhibiting ovarian aging. Therefore, we suggest that hAMSCs improve ovarian function in natural aging by secreting HGF and EGF.

Background RNA modification‐induced ovarian dysgenesis appears to be necessary for ovary development. However, how m5C (5‐methylcytosine)‐coordinating modificatory transcripts are dynamically regulated during oogenesis, and ovarian development is unknown. The purpose of this study was to determine whether NOP2/Sun RNA methyltransferase 5 (Nsun5) deletion leads to suppression of ovarian function and arrest of embryonic development. The regulation of mRNA decay and stability by m5C modification is essential at multiple stages during the maternal‐to‐zygotic (MZT) transition. Methods Mouse ovaries and oocytes with Nsun5KO and the KGN cell line were subjected to m5C identification, alternative splicing analysis and protein expression. BS‐m5C‐seq, real‐time polymerase chain reaction, Western blot, immunofluorescence and actinomycin D treatment assays were used. In particular, BS‐m5C‐seq revealed a dynamic pattern of m5C sites and genes in the ovaries between Nsun5KO and WT mice at the 2‐month and 6‐month stages. Diverse bioinformatic tools were employed to identify target genes for Nsun5. Results Here, a maternal mRNA stability study showed that deletion of the m5C methyltransferase Nsun5 obstructs follicular development and ovarian function, which leads directly to inhibition of embryogenesis and embryo development. Dynamic analysis of m5C revealed that the level of m5C decreased in a time‐dependent manner after Nsun5 knockout. Regarding the molecular mechanism, we found that Nsun5 deficiency caused a m5C decline in the exon and 3′UTR regions that influenced the translation efficiency of Mitotic arrest deficient 2 like 2 (MAD2L2) and Growth differentiation factor 9 (GDF9) in the ovary. Mechanistic investigation of alternative splicing indicated that Nsun5KO triggers aberrant events in the exon region of Brd8. Conclusions Nsun5 loss arrests follicular genesis and development in ovarian aging, indicating that Nsun5/m5C‐regulated maternal mRNA stabilization is essential for MZT transition. 1. Loss of Nsun5 affects Maternal mRNA stability 2. Nsun5 deficiency influences the translation efficiency of Mad2l2 and Gdf9 3. Nsun5 knockout triggers aberrant events in the exon region of Brd8