M6A demethylase FTO-stabilized exosomal circBRCA1 alleviates oxidative stress-induced granulosa cell damage via the miR-642a-5p/FOXO1 axis.

BACKGROUND: Premature ovarian insufficiency (POI) is an important cause of female infertility and seriously impacts the physical and psychological health of patients. Human umbilical cord mesenchymal stem cell-derived exosomes (HucMSCs-Exs, H-Exs) have exhibited protective effects on ovarian function with unclear mechanisms. METHODS: A comprehensive analysis of the Gene Expression Omnibus (GEO) database were used to identify POI-associated circRNAs and miRNAs. The relationship between HucMSC-derived exosomal circBRCA1/miR-642a-5p/FOXO1 axis and POI was examined by RT-qPCR, Western blotting, reactive oxygen species (ROS) staining, senescence-associated ß-gal (SA-ß-gal) staining, JC-1 staining, TEM, oxygen consumption rate (OCR) measurements and ATP assay in vivo and in vitro. RT-qPCR detected the expression of circBRCA1 in GCs and serum of patients with normal ovarian reserve function (n = 50) and patients with POI (n = 50); then, the correlation of circBRCA1 with ovarian reserve function indexes was analyzed. RESULTS: Herein, we found that circBRCA1 was decreased in the serum and ovarian granulosa cells (GCs) of patients with POI and was associated with decreased ovarian reserve. H-Exs improved the disorder of the estrous cycles and reproductive hormone levels, reduced the number of atretic follicles, and alleviated the apoptosis and senescence of GCs in rats with POI. Moreover, H-Exs mitigated mitochondrial damage and reversed the reduced circBRCA1 expression induced by oxidative stress in GCs. Mechanistically, FTO served as an eraser to increase the stability and expression of circBRCA1 by mediating the m6A demethylation of circBRCA1, and exosomal circBRCA1 sponged miR-642a-5p to block its interaction with FOXO1. CircBRCA1 insufficiency aggravated mitochondrial dysfunction, mimicking FTO or FOXO1 depletion effects, which was counteracted by miR-642a-5p inhibition. CONCLUSION: H-Exs secreted circBRCA1 regulated by m6A modification, directly sponged miR-642a-5p to upregulate FOXO1, resisted oxidative stress injuries in GCs and protected ovarian function in rats with POI. Exosomal circBRCA1 supplementation may be a general prospect for the prevention and treatment of POI.

Perimenopause Decreases SERCA2a Activity in the Hearts of a Mouse Model of Ovarian Failure.

Risk of cardiovascular disease mortality rises in women after menopause. While increased cardiovascular risk is largely attributed to postmenopausal declines in estrogens, the molecular changes in the heart that contribute to risk are poorly understood. Disruptions in intracellular calcium handling develop in ovariectomized mice and have been implicated in cardiac dysfunction. Using a mouse model of menopause in which ovarian failure occurs over 120 days, we sought to determine if perimenopause impacted calcium removal mechanisms in the heart and identify the molecular mechanisms. Mice were injected with 4-vinylcyclohexene diepoxide (VCD) to induce ovarian failure over 120 days, mimicking perimenopause. Hearts were removed at 60 and 120 days after VCD injections, representing the middle and end of perimenopause. SERCA2a function was significantly diminished at the end of perimenopause. Neither SERCA2a nor phospholamban expression changed at either time point, but phospholamban phosphorylation at S16 and T17 was dynamically altered. Intrinsic SERCA inhibitors sarcolipin and myoregulin increased >4-fold at day 60, as did the native activator DWORF. At the end of perimenopause, sarcolipin and myoregulin returned to baseline levels while DWORF was significantly reduced below controls. Sodium-calcium exchanger expression was significantly increased at the end of perimenopause. These results show that the foundation for increased cardiovascular disease mortality develops in the heart during perimenopause and that regulators of calcium handling exhibit significant fluctuations over time. Understanding the temporal development of cardiovascular risk associated with menopause and the underlying mechanisms is critical to developing interventions that mitigate the rise in cardiovascular mortality that arises after menopause.

[Effect of electroacupuncture on autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on the autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency (POI), and explore the mechanism of EA in improving POI. METHODS: Thirty-two female SD rats were randomly divided into a blank group (n=8) and a model making group (n=24). The rats in the model making group were injected intraperitoneally with cyclophosphamide for 15 days to establish the POI model (the dosage on the 1st day was 50 mg/kg, and 8 mg/kg from the 2nd day to 15th day). The successfully modeled rats were then randomly divided into a model group, an EA group, and an estradiol (E2) group, with 8 rats in each group. Rats in the EA group received EA at bilateral "Gongsun" (SP 4) with continuous wave, frequency of 2 Hz, and current intensity of 0.1 to 1 mA, 20 min per treatment, once daily for 14 days. Rats in the E2 group were administered with E2 (0.01 mg/mL) by gavage (10 mL/kg), once daily for 14 days. The changes in estrous cycle were observed by rapid Giemsa staining before and after modeling. After intervention, ovarian tissue morphology was observed by HE staining; serum levels of follicle-stimulating hormone (FSH), E2, anti-Mullerian hormone (AMH), and inhibin B (INHB) were detected by ELISA; immunofluorescence staining was used to observe the expression of p62 in ovarian granulosa cells; the ultrastructure of ovarian granulosa cells was observed by transmission electron microscopy, and the number of autophagosomes and autolysosomes was compared; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of p62, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) in ovarian tissue. RESULTS: The results of vaginal smears in the blank group showed regular cyclical changes; the rats in the model group showed prolonged estrous cycle or cycle arrest, mostly in proestrus or metestrus, with overall ovarian atrophy, disordered structure, and decreased granulosa cells. Compared with the blank group, rats in the model group showed increased serum FSH level (P<0.01), decreased serum levels of E2, AMH, and INHB (P<0.01), decreased positive expression of p62 in ovarian granulosa cells (P<0.01), with obvious swelling of ovarian granulosa cells, mild to moderate swelling of mitochondria, slight expansion of rough endoplasmic reticulum, and hypertrophy of Golgi apparatus; the number of autophagosomes and autolysosomes in the ovaries was increased (P<0.01), the expression of p62 protein and mRNA was decreased (P<0.01), and the expression of Beclin-1 and LC3 protein and mRNA in ovarian tissue was increased (P<0.01). Compared with the model group, rats in the EA group and the E2 group showed decreased serum FSH levels (P<0.01), increased levels of E2, AMH, and INHB (P<0.01), increased positive expression of p62 in ovarian granulosa cells (P<0.01), alleviated degree of ovarian granulosa cell damage, with relatively intact organelle morphology, and decreased number of autophagosomes and autolysosomes in the ovaries (P<0.01); the rats also showed increased expression of p62 protein and mRNA (P<0.01), and decreased expression of Beclin-1 and LC3 protein and mRNA (P<0.01) in ovarian tissue. CONCLUSION: EA at "Gongsun" (SP 4) could improve ovarian reserve function in POI rats by reducing the number of autophagosomes and autolysosomes, up-regulating p62 expression, and down-regulating Beclin-1 and LC3 expression, thus inhibiting autophagy of ovarian granulosa cells, and regulating the serum levels of FSH, E2, AMH, and INHB.

Network pharmacology and transcriptomics analysis reveal the mechanism of BushenHuoxue formula attenuates premature ovarian failure via modulation PI3K/AKT pathway.

This study aims to analyze the active components and mechanism of Bushen Huoxue (BSHX) formula on the autoimmune premature ovarian insufficiency (POI) by combining network pharmacology and Transcriptomics. The active components and targets of BSHXF were screened through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). POI-related targets were identified through Therapeutic Targets Database (TTD), DisGeNET and drugbank database. The Veen diagram was performed to obtain the action targets. The active compound-target network and Protein-Protein Interaction (PPI) network were built by using STRING database and Cytoscape software. Key targets and active compounds were further identified by topological analysis. Molecular docking shows that Kaempferol, Isorhamnetin and Anhydroicaritin have strong binding to AKT. Finally, a zp3-induced autoimmune ovarian function deficiency mouse model was used to explore the potential mechanism of POI. The potential pathways of BSHXF for the treatment of POI were identified by Transcriptomic analysis. PI3K-AKT and NF-kb pathways were the common pathways between network pharmacology and transcriptomics. Our results revealed that BSHXF could reduce the FSH expression levels and raise the E2, and AMH levels in the serum. Western bloting demonstrates that BSHXF could upregulate the expression of p-PI3K and p-AKT.

Moxibustion ameliorates ovarian function in premature ovarian insufficiency rats by activating cAMP/PKA/CREB to promote steroidogenesis in ovarian granulosa cells.

Premature ovarian insufficiency (POI) presents a substantial challenge to women's physiological and psychological well-being. Hormone replacement therapy, as the preferred therapeutic approach, involves solely exogenous supplementation of estrogen. Moxibustion, a traditional Chinese external treatment, has been investigated in our previous studies. It not only improves hormone levels and clinical symptoms in POI patients but also safeguards ovarian reserve. This study aims to explore the regulatory mechanisms by which moxibustion modulates hormone levels and restores ovarian function in POI. A POI rat model was established using cyclophosphamide, and moxibustion treatment was applied at acupoints "CV4" and "SP6" for a total of four courses. Subsequently, ovaries from each group were subjected to transcriptome sequencing (Bulk RNA-seq). Target pathways and key genes were selected through enrichment analysis and GSVA scoring, with validation using various techniques including electron microscopy, ELISA, Western blot, and immunohistochemistry. The results demonstrated that moxibustion restored the estrous cycle in POI rats, improved sex hormone levels, reduced the number of atretic follicles, and increased the count of dominant follicles (P<0.05). Bulk RNA-seq analysis revealed that moxibustion downregulated pathways associated with ovarian dysfunction, infertility, and immune responses, upregulated pathways related to follicular development and ovarian steroidogenesis. Furthermore, our data confirmed that moxibustion significantly increased the number of ovarian granulosa cells (GCs) and upregulated the expression of proteins related to steroidogenesis in GCs, including FSHR, P450 arom, cAMP, PKA, and CREB (P<0.05), with no significant effect observed on proteins related to steroidogenesis in theca cells. These outcomes aligned with the RNA-seq results. In conclusion, these findings propose that moxibustion enhances steroidogenesis in GCs through the activation of the cAMP/PKA/CREB pathway, consequently improving impaired ovarian function in POI rats. This study provides robust evidence supporting moxibustion as a targeted intervention for treating POI by specifically regulating steroidogenesis in GCs.

Non-targeted metabolomics revealed novel links between serum metabolites and primary ovarian insufficiency: a Mendelian randomization study.

Background: Primary ovarian insufficiency (POI) is a common clinical endocrine disorder with a high heterogeneity in both endocrine hormones and etiological phenotypes. However, the etiology of POI remains unclear. Herein, we unraveled the causality of genetically determined metabolites (GDMs) on POI through Mendelian randomization (MR) study with the overarching goal of disclosing underlying mechanisms. Methods: Genetic links with 486 metabolites were retrieved from GWAS data of 7824 European participants as exposures, while GWAS data concerning POI were utilized as the outcome. Via MR analysis, we selected inverse-variance weighted (IVW) method for primary analysis and several additional MR methods (MR-Egger, weighted median, and MR-PRESSO) for sensitivity analyses. MR-Egger intercept and Cochran's Q statistical analysis were conducted to assess potential heterogeneity and pleiotropy. In addition, genetic variations in the key target metabolite were scrutinized further. We conducted replication, meta-analysis, and linkage disequilibrium score regression (LDSC) to reinforce our findings. The MR Steiger test and reverse MR analysis were utilized to assess the robustness of genetic directionality. Furthermore, to deeply explore causality, we performed colocalization analysis and metabolic pathway analysis. Results: Via IVW methods, our study identified 33 metabolites that might exert a causal effect on POI development. X-11437 showed a robustly significant relationship with POI in four MR analysis methods (P IVW=0.0119; P weighted-median =0.0145; PMR-Egger =0.0499; PMR-PRESSO =0.0248). Among the identified metabolites, N-acetylalanine emerged as the most significant in the primary MR analysis using IVW method, reinforcing its pivotal status as a serum biomarker indicative of an elevated POI risk with the most notable P-value (P IVW=0.0007; PMR-PRESSO =0.0022). Multiple analyses were implemented to further demonstrate the reliability and stability of our deduction of causality. Reverse MR analysis did not provide evidence for the causal effects of POI on 33 metabolites. Colocalization analysis revealed that some causal associations between metabolites and POI might be driven by shared genetic variants. Conclusion: By incorporating genomics with metabolomics, this study sought to offer a comprehensive analysis in causal impact of serum metabolome phenotypes on risks of POI with implications for underlying mechanisms, disease screening and prevention.

Exosomal YB-1 facilitates ovarian restoration by MALAT1/miR-211-5p/FOXO3 axis.

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. Mesenchymal stem cells-derived small extracellular vesicles (MSCs-sEVs) are attractive candidates for ovarian function restoration and folliculogenesis for POF due to their safety and efficacy, however, the key mediator in MSCs-sEVs that modulates this response and underlying mechanisms remains elusive. Herein, we reported that YB-1 protein was markedly downregulated in vitro and in vivo models of POF induced with H2O2 and CTX respectively, accompanied by granulosa cells (GCs) senescence phenotype. Notably, BMSCs-sEVs transplantation upregulated YB-1, attenuated oxidative damage-induced cellular senescence in GCs, and significantly improved the ovarian function of POF rats, but that was reversed by YB-1 depletion. Moreover, YB-1 showed an obvious decline in serum and GCs in POF patients. Mechanistically, YB-1 as an RNA-binding protein (RBP) physically interacted with a long non-coding RNA, MALAT1, and increased its stability, further, MALAT1 acted as a competing endogenous RNA (ceRNA) to elevate FOXO3 levels by sequestering miR-211-5p to prevent its degradation, leading to repair of ovarian function. In summary, we demonstrated that BMSCs-sEVs improve ovarian function by releasing YB-1, which mediates MALAT1/miR-211-5p/FOXO3 axis regulation, providing a possible therapeutic target for patients with POF.

FGF2 promotes the proliferation of injured granulosa cells in premature ovarian failure via Hippo-YAP signaling pathway.

Young women undergoing anticancer treatment are at risk of premature ovarian failure (POF). Endometrial-derived stem cells (EnSCs) have demonstrated significant therapeutic potential for treating ovarian insufficiency, although the underlying mechanisms remain to be fully understood. This study aims to further investigate the therapeutic effects of EnSCs, particularly through the paracrine action of fibroblast growth factor 2 (FGF2), on POF. The findings show that exogenous FGF2 enhances the survival of ovarian granulosa cells damaged by cisplatin. FGF2 stimulates the proliferation of these damaged cells by suppressing the Hippo signaling pathway and activating YAP expression. In vivo experiments also revealed that FGF2 treatment significantly improves ovarian reserve and endocrine function in mice with POF. These results suggest that FGF2 can boost the proliferative capacity of damaged ovarian granulosa cells through the Hippo-YAP signaling pathway, providing a theoretical foundation for using EnSCs and FGF2 in clinical treatments for POF.

Exosomes derived from mesenchymal stem cells attenuate NLRP3-related pyroptosis in autoimmune premature ovarian insufficiency via the NF-κB pathway.

RESEARCH QUESTION: What is the effect of exosomes derived from bone marrow mesenchymal stem cells (MSC-Exos) on the pyroptosis and recovery of granulosa cells in autoimmune premature ovarian insufficiency (POI)? DESIGN: In vitro, KGN cells were exposed to interferon-gamma to simulate immune injury. Samples were collected after a 48 h incubation with MSC-Exos (30 µg/ml). The cell viability, secretion of oestrogen and expression of key molecules in pyroptosis and the nuclear factor kappa B (NF-κB) pathway were tested. In vivo, the BALB/c mouse model of autoimmune POI model induced by zona pellucida glycoprotein 3 was used. Fertility testing and sample collection were applied 4 weeks after the ovarian subcapsular injection of MSC-Exos (150 µg for each ovary). Hormone concentration measurements, follicle counting and pyroptotic pathway analyses were conducted for each group. RESULTS: In vitro, MSC-Exos significantly promoted the proliferation rate and secretion of oestrogen, while at the same time suppressing apoptosis and pyroptosis. In vivo, exosomal treatment normalized the irregular oestrous cycles, rescued the follicular loss and increased the pregnancy rate and number of offspring in POI mice. Elevated serum concentrations of oestrogen and anti-Müllerian hormone, as well as decreased concentrations of FSH and interleukin-1ß, were shown. Furthermore, MSC-Exos down-regulated the expression of the NLRP3/Casp1/GSDMD pathway and inhibited activation of the NF-κB pathway. CONCLUSIONS: These findings demonstrate for the first time that MSC-Exos exert a significant effect on restoring ovarian function in autoimmune POI in vivo and in vitro by suppressing the NLRP3/Casp1/GSDMD pathway and pyroptosis. The NF-κB pathway may contribute to the regulation of NLRP3-related pyroptosis.

Spatiotemporal transcriptomic changes of human ovarian aging and the regulatory role of FOXP1.

Limited understanding exists regarding how aging impacts the cellular and molecular aspects of the human ovary. This study combines single-cell RNA sequencing and spatial transcriptomics to systematically characterize human ovarian aging. Spatiotemporal molecular signatures of the eight types of ovarian cells during aging are observed. An analysis of age-associated changes in gene expression reveals that DNA damage response may be a key biological pathway in oocyte aging. Three granulosa cells subtypes and five theca and stromal cells subtypes, as well as their spatiotemporal transcriptomics changes during aging, are identified. FOXP1 emerges as a regulator of ovarian aging, declining with age and inhibiting CDKN1A transcription. Silencing FOXP1 results in premature ovarian insufficiency in mice. These findings offer a comprehensive understanding of spatiotemporal variability in human ovarian aging, aiding the prioritization of potential diagnostic biomarkers and therapeutic strategies.

Yu Linzhu alleviates primary ovarian insufficiency in a rat model by improving proliferation and energy metabolism of granulosa cells through hif1α/cx43 pathway.

BACKGROUND: Yu Linzhu (YLZ) is a classical Chinese traditional formula, which has been used for more than 600 years to regulate menstruation to help pregnancy. However, the mechanism of modern scientific action of YLZ needs to be further studied. METHODS: Thirty SD female rats were divided into three groups to prepare the blank serum and drug-containing serum, and then using UHPLC-QE-MS to identify the ingredients of YLZ and its drug-containing serum. Twenty-four SD female rats were divided into four groups, except the control group, 4-vinylcyclohexene dicycloxide (VCD) was intraperitoneally injected to establish a primary ovarian insufficiency (POI) model of all groups. Using vaginal smear to show that the estrous cycle of rats was disturbed after modeling, indicates that the POI model was successfully established. The ELISA test was used to measure the follicle-stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH) levels in the serum of rats. HE stain was used to assess the morphology of ovarian tissue. The localization and relative expression levels of CX43 protein were detected by tissue immunofluorescence. Primary ovarian granulosa cells (GCs) were identified by cellular immunofluorescence. CCK8 was used to screen time and concentration of drug-containing serum and evaluate the proliferation effect of YLZ on VCD-induced GCs. ATP kit and Seahorse XFe24 were used to detect energy production and real-time glycolytic metabolism rate of GCs. mRNA and protein expression levels of HIF1α, CX43, PEK, LDH, HK1 were detected by RT-PCR and WB. RESULTS: UHPLC-QE-MS found 1702 ingredients of YLZ and 80 constituents migrating to blood. YLZ reduced the FSH while increasing the AMH and E2 levels. In ovarian tissues, YLZ improved ovarian morphology, follicle development, and the relative expression of CX43. In vitro studies, we found that YLZ increased the proliferative activity of GCs, ATP levels, glycolytic metabolic rate, HIF1α, CX43, PEK, HK1, LDH mRNA, and protein levels. CONCLUSIONS: The study indicated that YLZ increased the proliferation and glycolytic energy metabolism of GCs to improve follicular development further alleviating ovarian function.

Human umbilical cord mesenchymal stem cells alleviate chemotherapy-induced premature ovarian insufficiency mouse model by suppressing ferritinophagy-mediated ferroptosis in granulosa cells.

Primary ovarian insufficiency (POI) in younger women (under 40) manifests as irregular periods, high follicle-stimulating hormone (FSH), and low estradiol (E2), often triggered by chemotherapy. Though mesenchymal stem cell (MSC) therapy shows promise in treating POI, its exact mechanism remains unclear. This study reveals that human umbilical cord-derived MSCs (hUC-MSCs) can protect ovarian granulosa cells (GCs) from cyclophosphamide (CTX)-induced ferroptosis, a form of cell death driven by iron accumulation. CTX, commonly used to induce POI animal model, triggered ferroptosis in GCs, while hUC-MSCs treatment mitigated this effect, both in vivo and in vitro. Further investigations using ferroptosis and autophagy inhibitors suggest that hUC-MSCs act by suppressing ferroptosis in GCs. Interestingly, hUC-MSCs activate a protective antioxidant pathway in GCs via NRF2, a stress-response regulator. Overall, our findings suggest that hUC-MSCs improve ovarian function in CTX-induced POI by reducing ferroptosis in GCs. This study not only clarifies the mechanism behind the benefits of hUC-MSCs but also strengthens the case for their clinical use in treating POI. Additionally, it opens up a new avenue for protecting ovaries from chemotherapy-induced damage by regulating ferroptosis.

miR-21-5p-loaded bone mesenchymal stem cell-derived exosomes repair ovarian function in autoimmune premature ovarian insufficiency by targeting MSX1.

RESEARCH QUESTION: What is the effect of micro-RNA (miR)-21-5p-loaded bone marrow mesenchymal stem cell-derived exosomes (miR-21-Exo) on autoimmune premature ovarian insufficiency (POI)? DESIGN: The Cell Counting Kit 8 (CCK8) assay, fluorescence-activated cell sorting, western blotting, quantitative reverse transcriptase (qRT)-PCR and enzyme-linked immunosorbent assay (ELISA) verified the effect of miR-21-Exo on interferon-γ (IFN-γ)-induced KGN cells. qRT-PCR, western blotting and dual-luciferase reporter gene assays verified that miR-21-Exo mediated Msh homeobox 1 (MSX1) regulation of the Notch signalling pathway and that miR-21 interacted directly with MSX1. The effects of miR-21-Exo on the ovaries were verified by monitoring of the oestrous cycle, haematoxylin and eosin staining, follicle counts, ELISA, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), western blotting and qRT-PCR. RESULTS: The results showed that miR-21-Exo promoted IFN-γ-induced KGN cell proliferation and hormone synthesis, and inhibited apoptosis. Using dual-luciferase reporter gene assays, miR-21 and MSX1 were shown to have direct interactions. Moreover, the findings elucidated that miR-21-Exo inhibited cell apoptosis and promoted hormone synthesis by mediating MSX1 to regulate the Notch signalling pathway. miR-21-Exo restored the ovarian structure in a mouse model of autoimmune POI, promoted endocrine function and proliferation, and inhibited apoptosis and inflammation in vivo. CONCLUSIONS: This study demonstrates that miR-21-Exo regulates the MSX1-mediated Notch signalling pathway to inhibit granulosa cell apoptosis and improve hormone synthesis function, providing insight into a potential mechanism of molecular therapy for the treatment of autoimmune POI.

The remodeling of ovarian function: targeted delivery strategies for mesenchymal stem cells and their derived extracellular vesicles.

Premature ovarian insufficiency (POI) is an essential cause of reduced fertility and quality of life in young women. Mesenchymal stem cells (MSCs) and MSCs-derived extracellular vesicles (EVs) have the ability to migrate to damaged tissues and are considered as promising therapeutic approaches for POI. However, the homing ability and therapeutic efficacy of MSCs administered in vivo are still insufficient, and their potential tumorigenicity and multi-differentiation potential also bring many doubts about their safety. The targeting ability and migration efficiency of MSCs can be improved by genetic engineering and surface modification, thereby maximizing their therapeutic efficacy. However, the use of viral vectors also has increased safety concerns. In addition, EVs, which seem to be the current therapeutic alternative to MSCs, are still poorly targeted for distribution, although they have improved in terms of safety. This paper reviews the comparative therapeutic effects of MSCs and their derived EVs on POI, their biodistribution after in vivo administration, and the most important possible ovarian targeting strategies. Difficulties such as homogeneity and yield before clinical application are also discussed. This article will provide new insights into precision therapy and targeted drug delivery for female ovarian diseases.

TP63 truncating mutation causes increased cell apoptosis and premature ovarian insufficiency by enhanced transcriptional activation of CLCA2.

BACKGROUND: Premature ovarian insufficiency (POI) is a severe disorder leading to female infertility. Genetic mutations are important factors causing POI. TP63-truncating mutation has been reported to cause POI by increasing germ cell apoptosis, however what factors mediate this apoptosis remains unclear. METHODS: Ninety-three patients with POI were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Whole-exome sequencing (WES) was performed for each patient. Sanger sequencing was used to confirm potential causative genetic variants. A minigene assay was performed to determine splicing effects of TP63 variants. A TP63-truncating plasmid was constructed. Real-time quantitative PCR, western blot analyses, dual luciferase reporter assays, immunofluorescence staining, and cell apoptosis assays were used to study the underlying mechanism of a TP63-truncating mutation causing POI. RESULTS: By WES of 93 sporadic patients with POI, we found a 14-bp deletion covering the splice site in the TP63 gene. A minigene assay demonstrated that the 14-bp deletion variant led to exon 13 skipping during TP63 mRNA splicing, resulting in the generation of a truncated TP63 protein (TP63-mut). Overexpression of TP63-mut accelerated cell apoptosis. Mechanistically, the TP63-mut protein could bind to the promoter region of CLCA2 and activate the transcription of CLCA2 several times compared to that of the TP63 wild-type protein. Silencing CLCA2 using a specific small interfering RNA (siRNA) or inhibiting the Ataxia Telangiectasia Mutated (ATM) pathway using the KU55933 inhibitor attenuated cell apoptosis caused by TP63-mut protein expression. CONCLUSION: Our findings revealed a crucial role for CLCA2 in mediating apoptosis in POI pathogenesis, and suggested that CLCA2 is a potential therapeutic target for POI.

Time course changes in in vivo muscle mechanical function and Ca2+ regulation of force following experimentally induced gradual ovarian failure in mice.

The abrupt cessation of ovarian hormone release is associated with declines in muscle contractile function, yet the impact of gradual ovarian failure on muscle contractility across peri-, early- and late-stage menopause remains unclear. In this study, a 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure mouse model was used to examine time course changes in muscle mechanical function. Plantar flexors of female mice (VCD: n = 10; CON: n = 8) were assessed at 40 (early perimenopause), 80 (late perimenopause), 120 (menopause onset) and 176 (late menopause) days post-initial VCD injection. A torque-frequency relationship was established across a range of frequencies (10-200 Hz). Isotonic dynamic contractions were elicited against relative loads (10-80% maximal isometric torque) to determine the torque-velocity-power relationship. Mice then performed a fatigue task using intermittent 100 Hz isometric contractions until torque dropped by 60%. Recovery of twitch, 10 Hz and 100 Hz torque were tracked for 10 min post-task failure. Additionally, intact muscle fibres from the flexor digitorum brevis underwent a fatigue task (50 repetitions at 70 Hz), and 10 and 100 Hz tetanic [Ca2+] were monitored for 10 min afterward. VCD mice exhibited 16% lower twitch torque than controls across all time points. Apart from twitch torque, 10 Hz torque and 10 Hz tetanic [Ca2+], where VCD showed greater values relative to pre-fatigue during recovery, no significant differences were observed between control and VCD mice during recovery. These results indicate that gradual ovarian failure has minimal detriments to in vivo muscle mechanical function, with minor alterations observed primarily for low-frequency stimulation during recovery from fatigue.

Gonadotropin upregulates intraovarian calpains-1 and -2 during ovarian follicular recruitment in the SD rat model.

Calpain role has been shown in the cumulus cell-oocyte complexes and, corpus luteum. We investigated the association of calpains-1 and -2 in ovarian folliculogenesis using the Sprague-Dawley (SD) rat model and steroidogenesis in the human granulosa cells (hGCs). We induced PCOS in 42-day-old SD rats by letrozole oral gavage for 21 days. Premature ovarian failure (POF) was induced in 21-day-old SD rats by 4-vinylcyclohexene diepoxide (VCD). Ovulation and ovarian hyperstimulatory (OHS) syndrome were induced by pregnant mare gonadotropin (PMSG) + human chorionic gonadotropin (hCG) treatments in 21 days SD rats, respectively. Steroidogenesis is stimulated in human granulosa cells (hGCs) by forskolin and the response of 17-beta-estradiol (E2) on calpains expression was checked in hGCs. The protein expression by immunoblotting and activity by biochemical assay of calpains-1 and -2 showed an oscillating pattern in the ovarian cycle. PMSG-induced follicular recruitment showed upregulation of calpains-1 and -2, but with no change during ovarian function cessation (POF). Upregulated calpain-2 expression and calpain activity was found in the hCG +PMSG-induced ovulation. Letrozole-induced PCOS showed downregulation of calpain-1, but upregulation of calpain-2. PMSG+hCG-induced OHS led to the upregulation of calpain-1. Letrozole and metformin separately increased the expression level of calpains-1 and -2 in the hGCs during luteinization. In conclusion, the expression levels of calpains -1 and -2 are increased with ovarian follicular recruitment by PMSG and calpain-1 is decreased in the PCOS condition, and letrozole and metformin upregulate the expression of calpains-1 and -2 during luteinization in the hGCs possibly via E2 action.

Metformin improves d-galactose induced premature ovarian insufficiency through PI3K-Akt-FOXO3a pathway.

PURPOSE: Metformin (MET), a first-line treatment for type 2 diabetes mellitus, restores ovarian function in women with polycystic ovary syndrome. MET has been shown to increase the rate of success for in vitro fertilization when utilized in assisted reproductive technologies. This study was designed to examine the impact of MET on ovarian function and fertility in a mouse model of galactose-induced premature ovarian insufficiency (POI). We further investigated the underlying mechanisms. MATERIALS AND METHODS: Female mice were divided into 4 groups: saline, d-galactose, d-galactose â€‹+ â€‹MET, and MET. Body weight, ovarian index, and fertility were assessed. The hormonal profile was done. Advanced glycation end products (AGEPs), receptor for advanced glycation end products (RAGE), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), forkhead box O3a (FOXO3a) expression were measured. Ovarian follicle counting and morphology were analyzed. Immunohistochemistry of cleaved caspase-3 expression was performed. RESULTS: Our findings demonstrated that MET reversed irregularities in the estrus cycle, enhanced the ovarian index, and improved the abnormal levels of hormones and AGEs induced by d-galactose. Furthermore, the expression levels of PI3K, Akt, FOXO3a, and RAGE were upregulated with d-galactose. However, MET attenuated their expression levels. The primordial follicles ratio was improved, whereas atretic follicles and apoptotic-related cleaved caspase-3 expression were decreased in the d-galactose â€‹+ â€‹MET group compared to the d-galactose group. CONCLUSION: This study demonstrates that MET partially rescued ovarian dysfunction and apoptosis induced by d-galactose via a mechanism involving PI3K-Akt-FOXO3a pathway. Our finding proposed that MET may be a promising alternative treatment for POI.

Loss of Calponin 2 causes premature ovarian insufficiency in mice.

BACKGROUND: Premature ovarian insufficiency (POI) is a condition defined as women developing menopause before 40 years old. These patients display low ovarian reserve at young age and difficulties to conceive even with assisted reproductive technology. The pathogenesis of ovarian insufficiency is not fully understood. Genetic factors may underlie most of the cases. Actin cytoskeleton plays a pivotal role in ovarian folliculogenesis. Calponin 2 encoded by the Cnn2 gene is an actin associated protein that regulates motility and mechanical signaling related cellular functions. RESULTS: The present study compared breeding of age-matched calponin 2 knockout (Cnn2-KO) and wild type (WT) mice and found that Cnn2-KO mothers had significantly smaller litter sizes. Ovaries from 4 weeks old Cnn2-KO mice showed significantly lower numbers of total ovarian follicles than WT control with the presence of multi-oocyte follicles. Cnn2-KO mice also showed age-progressive earlier depletion of ovarian follicles. Cnn2 expression is detected in the cumulus cells of the ovarian follicles of WT mice and colocalizes with actin stress fiber, tropomyosin and myosin II in primary cultures of cumulus cells. CONCLUSIONS: The findings demonstrate that the loss of calponin 2 impairs ovarian folliculogenesis with premature depletion of ovarian follicles. The role of calponin 2 in ovarian granulosa cells suggests a molecular target for further investigations on the pathogenesis of POI and for therapeutic development.

Gui ShenWan prevent premature ovarian insufficiency by modulating autophagy and angiogenesis via facilitating VDR.

ETHNOPHARMACOLOGICAL RELEVANCE: Gui Shen Wan (GSW) stands out as a promising therapeutic approach for addressing Premature Ovarian Insufficiency (POI). With deep roots in traditional medicine, GSW highlights the ethnopharmacological significance of herbal interventions in addressing nuanced aspects of women's health, with a specific emphasis on ovarian functionality. Recognizing the importance of GSW in gynecological contexts resonates with a rich tradition of using botanical formulations to navigate the intricacies of reproductive health. Delving into GSW's potential for treating POI emphasizes the crucial role of ethnopharmacological insights in guiding modern research endeavors. AIM OF THE STUDY: GSW is extensively utilized in gynecological disorders and has recently emerged as a potential therapeutic approach for POI. The present investigation aimed to assess the efficacy of GSW in treating POI in rats and elucidate its underlying molecular mechanisms. MATERIALS AND METHODS: The study employed GSW for POI treatment in rats. GSW, prepared as pills, underwent HPLC fingerprinting for quality control. Reagents and drugs, including VCD and dehydroepiandrosterone (DHEA), were sourced from reputable providers. Eighty Sprague-Dawley rats were categorized into groups for POI induction and treatment. Ovarian tissue underwent HE staining, immunohistochemical staining, Western Blot, qRT-PCR, and vaginal secretion testing. ELISA was utilized for target molecule detection. This methodology ensures a robust and reliable experimental framework. RESULTS: The results highlight a robust collaborative improvement in POI among rats subjected to combined GSW and DHEA treatment. Particularly noteworthy is the substantial enhancement in the expression of vascular regeneration-related molecules-VDR-Klotho-VEGFR-accompanied by a significant elevation in autophagy levels. Post-GSW administration, rat ovarian morphology demonstrated increased stability, hormone levels exhibited more consistent maintenance, and there was a marked reduction in inflammatory response compared to other groups (p < 0.01). Furthermore, GSW intervention resulted in a more pronounced upregulation of ovarian autophagy (p < 0.05). CONCLUSION: By modulating VDR-Klotho signaling, GSW exerts regulatory control over ovarian autophagy and vascular regeneration, thereby mitigating the occurrence and progression of POI in rats.