Fat mass and obesity-associated factor (FTO)-mediated N6-methyladenosine regulates spermatogenesis in an age-dependent manner.

N6-methyladenosine (m6A) is the most prevalent reversible RNA modification in the mammalian transcriptome. It has recently been demonstrated that m6A is crucial for male germline development. Fat mass and obesity-associated factor (FTO), a known m6A demethylase, is widely expressed in human and mouse tissues and is involved in manifold biological processes and human diseases. However, the function of FTO in spermatogenesis and male fertility remains poorly understood. Here, we generated an Fto knockout mouse model using CRISPR/Cas9-mediated genome editing techniques to address this knowledge gap. Remarkably, we found that loss of Fto in mice caused spermatogenesis defects in an age-dependent manner, resulting from the attenuated proliferation ability of undifferentiated spermatogonia and increased male germ cell apoptosis. Further research showed that FTO plays a vital role in the modulation of spermatogenesis and Leydig cell maturation by regulating the translation of the androgen receptor in an m6A-dependent manner. In addition, we identified two functional mutations of FTO in male infertility patients, resulting in truncated FTO protein and increased m6A modification in vitro. Our results highlight the crucial effects of FTO on spermatogonia and Leydig cells for the long-term maintenance of spermatogenesis and expand our understanding of the function of m6A in male fertility.

NSUN2-Mediated m5C Methylation Impairs Endometrial Receptivity.

Impaired endometrial decidualization is the primary cause of recurrent implantation failure (RIF). RNA methylation modification, especially NSUN family mediated m5C, is crucial for various physiological events, such as maternal-to-zygotic transition, gametogenesis, embryonic development, organismal lifespan, and cell cycle. However, the regulatory mechanisms between NSUN family mediated m5C modification and RIF remain unknown. We acquired NSUN2 expression data of 15 human endometrium samples at proliferative and secretory stages from reproductive cell atlas. The overall pattern of m5C sites and genes was elucidated through m5C-BS-seq, whereas the overall m5C levels in different groups were revealed by dot blot assay. BrdU and western blotting assays were carried out to evaluate the role of NSUN2 in proliferation and autophagy. The effects of NSUN2-mediated m5C modification on embryo attachment were evaluated by an in vitro model of a confluent monolayer of Ishikawa cells cocultured with BeWo spheroids, and its downstream targets were evaluated by real-time reverse-transcription PCR and western blotting in Ishikawa cells. The molecular mechanism for NSUN2 regulating its downstream targets' expression was determined by Cut&Tag and coimmunoprecipitation assays. NSUN2 was increased in SOX9+ cells and widespread in epithelial cell type at the proliferative stage by previous single-cell RNA sequencing data. NSUN2 overexpression (NSUN2OE) in the Ishikawa cell line elevated m5C levels and promoted cell proliferation and autophagy. NSUN2OE reduced attachment efficiency of BeWo cell spheres. Overexpressed NSUN2 was found to increase STAT1 and MMP14 mRNA expressions by inducing exon skipping. NSUN2 interacted with CLDN4 through m5C modification, and NSUN2OE or NSUN2 knockdown resulted in a similar variation tendency of CLDN4. Overexpression of NSUN2 increased CLDN4 H3K9ac modification by downregulating SIRT4 expression at the protein level, leading to the upregulation of CLDN4 mRNA expression. Our results uncovered a novel intricate regulatory mechanism between NSUN2-mediated m5C and RIF and suggested a potential new therapeutic strategy for RIF.

RNA methylation in neurodevelopment and related diseases.

Biological development and genetic information transfer are governed by genetic, epigenetic, transcriptional, and posttranscriptional mechanisms. RNA methylation, the attachment of methyl (-CH 3) groups to RNA molecules, is a posttranscriptional modification that has gained increasing attention in recent years because of its role in RNA epitranscriptomics. RNA modifications (RMs) influence various aspects of RNA metabolism and are involved in the regulation of diverse biological processes and diseases. Neural cell types emerge at specific stages of brain development, and recent studies have revealed that neurodevelopment, aging, and disease are tightly linked to transcriptome dysregulation. In this review, we discuss the roles of N6-methyladenine (m6A) and 5-methylcytidine (m5C) RNA modifications in neurodevelopment, physiological functions, and related diseases.

Exosomal miRNA-17-5p derived from human umbilical cord mesenchymal stem cells improves ovarian function in premature ovarian insufficiency by regulating SIRT7.

Premature ovarian insufficiency (POI) is clinically irreversible in women aged over 40 years. Although numerous studies have demonstrated satisfactory outcomes of mesenchymal stem cell therapy, the underlying therapeutic mechanism remains unclear. Exosomes were collected from the culture medium of human umbilical cord mesenchymal stem cells (hUMSCs) and assessed by electron microscopy and Western blot (WB) analysis. Then, exosomes were added to the culture medium of cyclophosphamide (CTX)-damaged human granulosa cells (hGCs), and the mixture was injected into the ovaries of CTX-induced POI model mice before detection of antiapoptotic and apoptotic gene expression. Next, the microRNA expression profiles of hUMSC-derived exosomes (hUMSC-Exos) were detected by small RNA sequencing. The ameliorative effect of exosomal microRNA-17-5P (miR-17-5P) was demonstrated by miR-17-5P knockdown before assessment of ovarian phenotype and function, reactive oxygen species (ROS) levels and SIRT7 expression. Finally, SIRT7 was inhibited or overexpressed by RNA interference or retrovirus transduction, and the protein expression of PARP1, γH2AX, and XRCC6 was analyzed. The ameliorative effect of hUMSC-Exos on POI was validated. Our results illustrated that hUMSC-Exos restored ovarian phenotype and function in a POI mouse model, promoted proliferation of CTX-damaged hGCs and ovarian cells, and alleviated ROS accumulation by delivering exosomal miR-17-5P and inhibiting SIRT7 expression. Moreover, our findings elucidated that miR-17-5P repressed PARP1, γH2AX, and XRCC6 by inhibiting SIRT7. Our findings suggest a critical role for exosomal miR-17-5P and its downstream target mRNA SIRT7 in hUMSC transplantation therapy. This study indicates the promise of exosome-based therapy for POI treatment.

Vitamin C- and Valproic Acid-Induced Fetal RPE Stem-like Cells Recover Retinal Degeneration via Regulating SOX2.

Retinal pigment epithelial (RPE) cell replacement therapy has provided promising outcomes in the treatment of retinal degenerative diseases (RDDs), but the resulting limited visual improvement has raised questions about graft survival and differentiation. Through combined treatment with vitamin C and valproic acid (together, VV), we activated human fetal RPE (fRPE) cells to become highly proliferative fetal RPE stem-like cells (fRPESCs). In this study, we report that SOX2 (SRY-box 2) activation contributed to mesenchymal-epithelial transition and elevated the retinal progenitor and mesenchymal stromal markers expressions of fRPESCs. These fRPESCs could differentiate into RPE cells, rod photoreceptors, and mesenchymal lineage progenies under defined conditions. Finally, fRPESCs were transplanted into the subretinal space of an RDD mouse model, and a photoreceptor rescue benefit was demonstrated. The RPE and rod photoreceptor differentiation of transplanted fRPESCs may account for the neural retinal recovery. This study establishes fRPESCs as a highly proliferative, multi-lineage differentiation potential (including RPE, rod photoreceptor, and mesenchymal lineage differentiation), mesenchymal-to-epithelial-transitioned retinal stem-like cell source for cell-based therapy of RDDs.

Stress, anxiety, and depression in infertile couples are not associated with a first IVF or ICSI treatment outcome.

BACKGROUND: Psychological distress may exert a negative influence on reproductive function of couples at reproductive age. Couples seeking assisted reproductive technology (ART) treatment may have a higher prevalence of psychological distress than fertile couples. However, whether psychological distress is associated with the outcome of ART treatment remains unknown. We aimed to investigate the association of pre-treatment psychological distress and clinical pregnancy rate among infertility couples undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment. METHODS: This nested case-control study was conducted based on women who underwent their first fresh IVF or ICSI cycle in the Jiangsu Birth Cohort Study (JBC) between November 2015 and January 2019. A total of 150 women who did not obtain clinical pregnancy after first IVF or ICSI fresh embryo transfer were identified as cases, and a total of 300 age matched women who obtained clinical pregnancy were identified as controls. Conditional logistic regression analyses were used to investigate the association between psychological distress and the outcome of first IVF or ICSI treatment, adjusting for multiple potential confounders. RESULTS: No statistically significant association was observed between score of maternal symptoms of psychological distress and clinical pregnancy. Adjusted ORs of logistic regression were 1.00 (95% CI 0.97-1.03) for anxiety, 0.98 (95% CI 0.95-1.02) for depression, and 0.98 (95% CI 0.95-1.01) for perceived stress, respectively. When treat depression and anxiety as categorical variables, 62 (13.8%) were classified as clinical depression, 11 (2.4%) were classified as clinical anxiety, among 450 women in the present study. Psychological distress symptoms were also not associated with clinical pregnancy rate. Adjusted ORs of logistic regression were 0.27 (95% CI 0.03-2.33) for anxiety, 0.88 (95% CI 0.46-1.68) for depression, respectively. CONCLUSIONS: Our findings firstly indicated that psychological distress experienced prior to IVF/ICSI treatment was not associated with clinical pregnancy.

Increased N6-methyladenosine causes infertility is associated with FTO expression.

The N6-methyladenosine (m6A) modification plays a central role in epigenetic regulation of the mammalian transcriptome. m6A can be demethylated by the fat mass- and obesity-associated (FTO) protein and the α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) protein. Much less is known about that whether m6A content is involved in POI (premature ovarian insufficiency) disease. In this case-controlled study, 69 POI and 53 tubal occlusion patients were recruited from the reproduction centers in our hospital. For the POI animal model experiment, ovarian tissue was obtained from ten POI and nine healthy mice. An m6A test kit was developed to determine the m6A content in the RNA, and qPCR and western blot were used to examine the mRNA and protein expression levels of FTO and ALKBH5. FACS was used to measure the levels of proliferation and apoptosis, and siRNA was used to establish FTO and ALKBH5 knockdown cell lines. Our results showed that the m6A content in the RNA from POI patients and POI mice was significantly higher than control groups and that POI was characterized by the content of m6A. The mRNA and protein expression levels of FTO were significantly lower in the POI patients than control group and were associated with a risk of POI. These data suggest that the decreased mRNA and protein expression levels of FTO may be responsible for the increase in m6A in POI, which may further increase the risk of complications of POI. High m6A should be investigated further as a novel potential biomarker of POI.

HGF and BFGF Secretion by Human Adipose-Derived Stem Cells Improves Ovarian Function During Natural Aging via Activation of the SIRT1/FOXO1 Signaling Pathway.

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.

17ß-Oestradiol promotes differentiation of human embryonic stem cells into dopamine neurons via cross-talk between insulin-like growth factors-1 and oestrogen receptor ß.

Human embryonic stem cells (hESCs) can self-renew and differentiate into all cell lineages. E2 is known to exhibit positive effects on embryo development. Although the importance of E2 in many physiological processes has been reported, to date few researchers have investigated the effects of E2 on hESCs differentiation. We studied the effects of E2 on dopamine (DA) neuron induction of hESCs and its related signalling pathways using the three-stage protocol. In our study, 0.1 µM E2 were applied to hESCs-derived human embryoid bodies (hEBs) and effects of E2 on neural cells differentiation were investigated. Protein and mRNA level assay indicated that E2 up-regulated the expression of insulin-like growth factors (IGF)-1, ectoderm, neural precursor cells (NPC) and DA neuron markers, respectively. The population of hESC-derived NPCs and DA neurons was increased to 92% and 93% to that of DMSO group, respectively. Furthermore, yield of DA neuron-secreted tyrosine hydroxylase (TH) and dopamine was also increased. E2-caused promotion was relieved in single inhibitor (ICI or JB1) group partly, and E2 effects were repressed more stronger in inhibitors combination (ICI plus JB1) group than in single inhibitor group at hEBs, hNPCs and hDA neurons stages. Owing to oestrogen receptors regulate multiple brain functions, when single or two inhibitors were used to treat neural differentiation stage, we found that oestrogen receptor (ER)ß but not ERα is strongly repressed at the hNPCs and hDA neurons stage. These findings, for the first time, demonstrate the molecular cascade and related cell biology events involved in E2-improved hNPC and hDA neuron differentiation through cross-talk between IGF-1 and ERß in vitro.

Protective role of stem cells in POI: Current status and mechanism of action, a review article.

Premature ovarian insufficiency (POI) has far-reaching consequences on women's life quality. Due to the lack of full recognition of the etiology and complexity of this disease, there is no appropriate treatment for infected patients. Recently, stem cell therapy has attracted the attention of regenerative medicine scholars and offered promising outcomes for POI patients. Several kinds of stem cells, such as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) have been used for the treatment of ovarian diseases. However, their potential protective mechanisms are still unknown. Undoubtedly, a better understanding of the therapeutic molecular and cellular mechanisms of stem cells will address uncover strategies to increase their clinical application for multiple disorders such as POI. This paper describes a detailed account of the potential properties of different types of stem cells and provides a comprehensive review of their protective mechanisms, particularly MSC, in POI disorder. In addition, ongoing challenges and several strategies to improve the efficacy of MSC in clinical use are addressed. Therefore, this review will provide proof-of-concept for further clinical application of stem cells in POI.

A low-cost, low-input method establishment for m6A MeRIP-seq.

N6-methyladenosine (m6A) is a highly prevalent modification found in mammal mRNA molecules that plays a crucial role in the regulation of cellular function. m6A RNA immunoprecipitation sequencing (MeRIP-seq) has been frequently used in transcriptomics research to identify the location of m6A. MABE572 (Millipore) is the most widely utilized and efficient anti-m6A antibody for MeRIP-seq. However, due to the high dose and price of this antibody, which has also been taken off the market, we discovered that CST's anti-m6A antibody can be used instead of MABE572 to map the m6A transcriptome. In the present study, we performed different concentrations of the CST anti-m6A antibodies with the corresponding initiation RNA of HEK293T cells, 2.5 µg antibody with 1 µg total RNA, 1.25 µg antibody with 0.5 µg total RNA, and 1.25 µg antibody with 0.1 µg total RNA. By comparing the m6A peak calling, enriched motifs, alternative splicing events, and nuclear transcripts modified by m6A between the CST and Millipore libraries, it was found that the CST library presented similar data to Millipore, even at incredibly low doses. The volume and cost of antibodies are significantly reduced by this refined MeRIP-seq using CST antibody, making it convenient to map future large-scale sample m6A methylation.

Expert Consensus on Prevention and Treatment of Aging-Related Gonadal Dysfunction.

Aging-related hypogonadism involves complex mechanisms in humans, predominantly relating to the decline of multiple hormones and senile gonads. Late-onset hypogonadism (LOH) and erectile dysfunction (ED) are the main manifestations in men, while premature ovarian insufficiency (POI) and menopause are the main forms in women. Anti-aging measures include lifestyle modification and resistance training, hormonal supplementation, stem cell therapy, metformin, and rapamycin. In this expert consensus, the mechanisms, efficacy, and side effects of stem cell therapy on aging gonadal function are reviewed. Furthermore, various methods of stem cell therapy, administered intravenously, intracavernously, and intra-ovarially, are exemplified in detail. More clinical trials on aging-related gonadal dysfunction are required to solidify the foundation of this topic.

Normal human embryonic stem cell lines were derived from microsurgical enucleated tripronuclear zygotes.

A normal fertilized human zygote contains two pronuclei, but zygotes may also display one, three, or even more pronuclei resulting from irregular insemination or meiotic division. Today diploid and triploid human embryonic stem cell (hESC) lines have been derived from tripronuclear (3PN) triploid zygotes, and an in-vitro fertilization (IVF) baby was born from a rescued diploid zygote by removing the extra male pronucleus of the 3PN zygote. However, whether hESCs can be derived from a rescued 3PN zygote is still unknown. Here, by microsurgical pronuclear removal, we restored 61 diploid zygotes from 3PN zygotes donated by 35 couples, and 11 blastocysts developed with a blastocyst rate of 18.0%, which seems higher than that of nonrescued 3PN zygotes according to previous reports. After the whole zona pellucida free embryos were plated onto feeder cells to grow and passage, 2 hESC lines (CCRM-hESC-22 and CCRM-hESC-23) were generated and both carried normal karyotype (46, XY). The hESC lines were then characterized by morphology, expansion in vitro, and expression of specific markers of alkaline phosphatase, OCT4, SSEA4, TRA-1-60 and TRA-1-81. Furthermore, the pluripotency of these 2 hESC lines was confirmed by in vitro embryoid body formation and in vivo teratoma production. Our study indicates that depronucleared 3PN zygotes can improve the blastocysts formation rate, and normal hESC lines can be derived from those corrected 2PN embryos. Based on their multi-directional differentiation potential in vitro, the established hESC lines could be applied to the developmental risk assessment for IVF babies born from restored zygotes.

The Role of m6A on Female Reproduction and Fertility: From Gonad Development to Ovarian Aging.

The growth and maturation of oocyte is accompanied by the accumulation of abundant RNAs and posttranscriptional regulation. N6-methyladenosine (m6A) 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 m6A 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 m6A 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 m6A 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 m6A modification involved in female reproduction and fertility is still unknown. In this review, we have summarized the current findings of the RNA m6A 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 m6A modification in promoting oocyte maturation and delaying the reproduction aging.

RNA-methyltransferase Nsun5 controls the maternal-to-zygotic transition by regulating maternal mRNA stability.

BACKGROUND: RNA modification-induced ovarian dysgenesis appears to be necessary for ovary development. However, how m5 C (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 m5 C 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 m5 C identification, alternative splicing analysis and protein expression. BS-m5 C-seq, real-time polymerase chain reaction, Western blot, immunofluorescence and actinomycin D treatment assays were used. In particular, BS-m5 C-seq revealed a dynamic pattern of m5 C 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 m5 C methyltransferase Nsun5 obstructs follicular development and ovarian function, which leads directly to inhibition of embryogenesis and embryo development. Dynamic analysis of m5 C revealed that the level of m5 C decreased in a time-dependent manner after Nsun5 knockout. Regarding the molecular mechanism, we found that Nsun5 deficiency caused a m5 C 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/m5 C-regulated maternal mRNA stabilization is essential for MZT transition.

FTO mediates LINE1 m6A demethylation and chromatin regulation in mESCs and mouse development.

N6-methyladenosine (m6A) is the most abundant internal modification on mammalian messenger RNA. It is installed by a writer complex and can be reversed by erasers such as the fat mass and obesity-associated protein FTO. Despite extensive research, the primary physiological substrates of FTO in mammalian tissues and development remain elusive. Here, we show that FTO mediates m6A demethylation of long-interspersed element-1 (LINE1) RNA in mouse embryonic stem cells (mESCs), regulating LINE1 RNA abundance and the local chromatin state, which in turn modulates the transcription of LINE1-containing genes. FTO-mediated LINE1 RNA m6A demethylation also plays regulatory roles in shaping chromatin state and gene expression during mouse oocyte and embryonic development. Our results suggest broad effects of LINE1 RNA m6A demethylation by FTO in mammals.

Concentrated exosomes from menstrual blood-derived stromal cells improves ovarian activity in a rat model of premature ovarian insufficiency.

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.

Human placental mesenchymal stem cells ameliorate chemotherapy-induced damage in the testis by reducing apoptosis/oxidative stress and promoting autophagy.

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.

Human Amniotic Fluid Mesenchymal Stem Cells Improve Ovarian Function During Physiological Aging by Resisting DNA Damage.

Many studies have shown that mesenchymal stem cells have the ability to restore function in models of premature ovarian insufficiency disease, but few studies have used stem cells in the treatment of ovarian physiologic aging (OPA). This experimental study was designed to determine whether human amniotic fluid mesenchymal stem cells (hAFMSCs) have the ability to recover ovarian vitality and to determine how they function in this process. Mice (12-14 months old) were used in this study, and young fertile female mice (3-5 months old) were the control group. Ovarian markers for four stages of folliculogenesis and DNA damage genes were tested by qPCR and western blot. hAFMSCs were used to treat an OPA mouse model, and the animals treated with hAFMSCs displayed better therapeutic activity in terms of the function of the mouse ovary, increasing follicle numbers and improving hormone levels. In addition, our results demonstrated that the marker expression level in ovarian granular cells from patients with OPA was elevated significantly after hAFMSC treatment. In addition, the proliferation activity was improved, and apoptosis was dramatically inhibited after hAFMSCs were cocultured with hGCs from OPA patients. Finally, in this study, hAFMSCs were shown to increase the mRNA and protein expression levels of ovarian markers at four stages of folliculogenesis and to inhibit the expression of DNA damage genes. These works have provided insight into the view that hAFMSCs play an integral role in resisting OPA. Moreover, our present study demonstrates that hAMSCs recover ovarian function in OPA by restoring the expression of DNA damage genes.

EGF released from human placental mesenchymal stem cells improves premature ovarian insufficiency via NRF2/HO-1 activation.

Human placental mesenchymal stem cells (hPMSCs) have the ability to release cytokines and to differentiate into the three germ layers. To date, the relevance of hPMSCs for the treatment of premature ovarian insufficiency (POI) disease through the regulation of oxidative stress is still unclear. Therefore, to evaluate the therapeutic efficiency and investigate the mechanism of hPMSCs, we generated a mouse model of POI and collected human ovarian granule cells (hGCs) from patients with POI. hPMSCs displayed therapeutic effects on POI ovarian function, including recovered follicular numbers and increased expression of oocyte markers. Furthermore, secretion of the cytokine EGF (epidermal growth factor) was higher from hPMSCs than it was from other cells. FACS and Western blot analyses showed that EGF elevated the proliferation and reduced the apoptosis in hGCs. hPMSCs and EGF inhibited oxidative stress levels. Protein assays demonstrated that EGF suppressed oxidative stress by dose-dependently upregulating the expression of the NRF2/HO-1 pathway, and it inhibited the apoptosis by regulating the PTEN/PI3K/AKT pathway. These findings provide an experimental foundation for hPMSCs in improving ovarian function through the secretion of EGF. The mechanism of action of EGF is related to protection from oxidative stress by activation of the NRF2/HO-1.