RESUMEN
BACKGROUND: Premature ovarian insufficiency (POI) is a condition characterized by a substantial decline or loss of ovarian function in women before the age of 40. However, the pathogenesis of POI remains to be further elucidated, and specific targeted drugs which could delay or reverse ovarian reserve decline are urgently needed. Abnormal DNA damage repair (DDR) and cell senescence in granulosa cells are pathogenic mechanisms of POI. Ubiquitin-specific protease 14 (USP14) is a key enzyme that regulates the deubiquitylation of DDR-related proteins, but whether USP14 participates in the pathogenesis of POI remains unclear. METHODS: We measured USP14 mRNA expression in granulosa cells from biochemical POI (bPOI) patients. In KGN cells, we used IU1 and siRNA-USP14 to specifically inhibit USP14 and constructed a cell line stably overexpressing USP14 to examine its effects on DDR function and cellular senescence in granulosa cells. Next, we explored the therapeutic potential of IU1 in POI mouse models induced by D-galactose. RESULTS: USP14 expression in the granulosa cells of bPOI patients was significantly upregulated. In KGN cells, IU1 treatment and siUSP14 transfection decreased etoposide-induced DNA damage levels, promoted DDR function, and inhibited cell senescence. USP14 overexpression increased DNA damage, impaired DDR function, and promoted cell senescence. Moreover, IU1 treatment and siUSP14 transfection increased nonhomologous end joining (NHEJ), upregulated RNF168, Ku70, and DDB1, and increased ubiquitinated DDB1 levels in KGN cells. Conversely, USP14 overexpression had the opposite effects. Intraperitoneal IU1 injection alleviated etoposide-induced DNA damage in granulosa cells, ameliorated the D-galactose-induced POI phenotype, promoted DDR, and inhibited cell senescence in ovarian granulosa cells in vivo. CONCLUSIONS: Upregulated USP14 in ovarian granulosa cells may play a role in POI pathogenesis, and targeting USP14 may be a potential POI treatment strategy. Our study provides new insights into the pathogenesis of POI and a novel POI treatment strategy.
Asunto(s)
Senescencia Celular , Daño del ADN , Reparación del ADN , Células de la Granulosa , Insuficiencia Ovárica Primaria , Ubiquitina Tiolesterasa , Femenino , Insuficiencia Ovárica Primaria/patología , Insuficiencia Ovárica Primaria/metabolismo , Insuficiencia Ovárica Primaria/genética , Células de la Granulosa/metabolismo , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/patología , Senescencia Celular/efectos de los fármacos , Animales , Humanos , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Reparación del ADN/efectos de los fármacos , Ratones , Adulto , Ratones Endogámicos C57BL , Línea CelularRESUMEN
Cis-dichlorodiammineplatinum(II) (CDDP), while widely utilized in tumor therapy, results in toxic side effects that patients find intolerable. The specific mechanism by which CDDP inflicts ovarian damage remains unclear. This study aimed to explore the involvement of ferrostatin-1 (FER-1) and ferroptosis in CDDP-induced ovarian toxicity. This study established models of CDDP-induced injury in granulosa cells (GCs) and rat model of premature ovarian failure (POF). CCK-8 assessed the effects of CDDP and FER-1 on GC viability. FerroOrange and Mito-FerroGreen, DCFH-DA and MitoSox-Red, Rhodamine 123 and Transmission electron microscopy (TEM) measured Fe2+, reactive oxygen species (ROS), mitochondrial membrane potential and the mitochondrial morphology in GC cells, respectively. Serum hormone levels; organ indices; malondialdehyde, superoxide dismutase, and glutathione analyses; and western blotting were performed to examine ferroptosis's role in vitro. Molecular docking simulation was evaluated the interaction between FER-1 and GPX4 or FER-1 and NRF2. Molecular docking simulations were conducted to evaluate the interactions between FER-1 and GPX4, as well as FER-1 and NRF2. The findings revealed that CDDP-induced ovarian toxicity involved iron accumulation, increased ROS accumulation, and mitochondrial dysfunction, leading to endocrine disruption and tissue damage in rats. These changes correlated with NRF2, HO-1, and GPX4 levels. However, FER-1 decreased the extent of ferroptosis. Thus, ferroptosis appears to be a crucial mechanism of CDDP-induced ovarian injury, with GPX4 as potential protective targets.
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Cisplatino , Ciclohexilaminas , Ferroptosis , Simulación del Acoplamiento Molecular , Fenilendiaminas , Especies Reactivas de Oxígeno , Animales , Femenino , Ferroptosis/efectos de los fármacos , Ciclohexilaminas/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Cisplatino/efectos adversos , Fenilendiaminas/farmacología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo , Ovario/patología , Insuficiencia Ovárica Primaria/inducido químicamente , Insuficiencia Ovárica Primaria/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratas Sprague-Dawley , Modelos Animales de Enfermedad , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismoRESUMEN
5-Hydroxytryptamine (5-HT) is an inhibitory neurotransmitter widely distributed in mammalian tissues, exerting its effects through binding to various receptors. It plays a crucial role in the proliferation of granulosa cells (GCs) and the development of follicles in female animals, however, its effect on porcine follicle development is not clear. The aim of this study is to investigate the expression of 5-HT and its receptors in various parts of the pig ovary, as well as the effect of 5-HT on porcine follicular development by using ELISA, quantitative real-time PCR (qPCR) and EdU assays. Firstly, we examined the levels of 5-HT and its receptors in porcine ovaries, follicles, and GCs. The findings revealed that the expression of different 5-HT receptors varied among follicles of different sizes. To investigate the relationship between 5-HT and its receptors, we exposed the GCs to 5-HT and found a decrease in 5-HT receptor expression compared to the control group. Subsequently, the treatment of GCs with 0.5 µM, 5 µM, and 50 µM 5-HT showed an increase in the expression of cell cycle-related genes, and EdU results indicated cell proliferation after the 0.5 µM 5-HT treatment. Additionally, the expression of genes involved in E2 synthesis was examined after the treatment of granulosa cells with 0.5 µM 5-HT. The results showed that CYP19A1 and HSP17ß1 expression was decreased. These results suggest that 5-HT might affect the development of porcine follicle by promoting the proliferation of GCs and inhibiting the synthesis of estrogen. This provides a new finding for exploring the effect of 5-HT on follicular development, and lays a foundation for further research on the mechanism of 5-HT in follicles.
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Proliferación Celular , Células de la Granulosa , Folículo Ovárico , Receptores de Serotonina , Serotonina , Animales , Serotonina/farmacología , Serotonina/metabolismo , Femenino , Porcinos , Folículo Ovárico/metabolismo , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/crecimiento & desarrollo , Células de la Granulosa/metabolismo , Células de la Granulosa/efectos de los fármacos , Receptores de Serotonina/metabolismo , Receptores de Serotonina/genética , Proliferación Celular/efectos de los fármacosRESUMEN
Background: Ovarian granulosa cells (GCs) are essential for follicular development. Ovarian advanced glycation end-products (AGEs) accumulation is related to GCs dysfunction. Alpha-lipoic acid (ALA) illustrates therapeutic capabilities for infertility-related disorders. Therefore, this study assessed the effects of ALA on AGEs-induced GCs hormonal dysfunction. Methods: The study was conducted from October 2021 to September 2022 at the Department of Medical Genetics, Shiraz University of Medical Sciences. Isolated GCs (n=50) were divided into control, human glycated albumin (HGA), HGA+ALA, and ALA treatments. Steroidogenic enzymes and AGE receptor (RAGE) genes were assessed by qRT-PCR. Steroid hormones and RAGE protein were evaluated using ELISA and Western blotting. Data were analyzed using GraphPad Prism software (ver. 9), and P<0.05 was considered significant. Results: Our findings showed that HGA treatment significantly (P=0.0001) increased RAGE (by 140.66%), STAR (by 117.65%), 3ß-HSD (by 165.68%), and 17ß-HSD (by 122.15%) expression, while it decreased CYP19A1 (by 68.37%) expression. RAGE protein level (by 267.10%) was also increased in HGA-treated GCs. A significant decrease in estradiol (by 59.66%) and a slight and sharp elevation in progesterone (by 30.40%) and total testosterone (by 158.24%) levels was also observed. ALA treatment ameliorated the HGA-induced changes in steroidogenic enzyme mRNA levels (P=0.001) and steroid hormone secretion (P=0.010). Conclusion: This work shows that ALA therapy likely corrects hormonal dysfunctions caused by AGEs in luteinized GCs. This effect is probably achieved by decreased RAGE expression. Clinical research is needed to understand how AGEs and ALA interact in the ovary, which might lead to a more targeted ovarian dysfunction therapy.
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Productos Finales de Glicación Avanzada , Células de la Granulosa , Ácido Tióctico , Humanos , Ácido Tióctico/farmacología , Ácido Tióctico/uso terapéutico , Células de la Granulosa/efectos de los fármacos , Femenino , Productos Finales de Glicación Avanzada/farmacología , Receptor para Productos Finales de Glicación Avanzada , Células CultivadasRESUMEN
The aim of the present study was to determine whether adipokines monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) can affect the functions of ovarian cells in cats. The addition of either MCP-1 or PAI-1 increased viability; promoted the accumulation of proliferation markers and progesterone and estradiol release; and decreased the accumulation of apoptosis markers in cultured feline granulosa cells. The present observations suggest that MCP-1 or PAI-1 can be physiological stimulators of ovarian granulosa cell functions.
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Quimiocina CCL2 , Células de la Granulosa , Inhibidor 1 de Activador Plasminogénico , Animales , Gatos , Femenino , Inhibidor 1 de Activador Plasminogénico/metabolismo , Células de la Granulosa/metabolismo , Células de la Granulosa/fisiología , Células de la Granulosa/efectos de los fármacos , Quimiocina CCL2/metabolismo , Células Cultivadas , Proliferación Celular/fisiología , Estradiol/metabolismo , Estradiol/farmacología , Progesterona/metabolismo , Progesterona/farmacología , Apoptosis , Supervivencia CelularRESUMEN
Salt-inducible kinases (SIKs), a family of serine/threonine kinases, were found to be critical determinants of female fertility. SIK2 silencing results in increased ovulatory response to gonadotropins. In contrast, SIK3 knockout results in infertility, gonadotropin insensitivity, and ovaries devoid of antral and preovulatory follicles. This study hypothesizes that SIK2 and SIK3 differentially regulate follicle growth and fertility via contrasting actions in the granulosa cells (GCs), the somatic cells of the follicle. Therefore, SIK2 or SIK3 GC-specific knockdown (SIK2GCKD and SIK3GCKD, respectively) mice were generated by crossing SIK floxed mice with Cyp19a1pII-Cre mice. Fertility studies revealed that pup accumulation over 6 months and the average litter size of SIK2GCKD mice were similar to controls, although in SIK3GCKD mice were significantly lower compared to controls. Compared to controls, gonadotropin stimulation of prepubertal SIK2GCKD mice resulted in significantly higher serum estradiol levels, whereas SIK3GCKD mice produced significantly less estradiol. Cyp11a1, Cyp19a1, and StAR were significantly increased in the GCs of gonadotropin-stimulated SIK2GCKD mice. However, Cyp11a1 and StAR remained significantly lower than controls in SIK3GCKD mice. Interestingly, Cyp19a1 stimulation in SIK3GCKD was not statistically different compared to controls. Superovulation resulted in SIK2GCKD mice ovulating significantly more oocytes, whereas SIK3GCKD mice ovulated significantly fewer oocytes than controls. Remarkably, SIK3GCKD superovulated ovaries contained significantly more preantral follicles than controls. SIK3GCKD ovaries contained significantly more apoptotic cells and fewer proliferating cells than controls. These data point to the differential regulation of GC function and follicle development by SIK2 and SIK3 and supports the therapeutic potential of targeting these kinases for treating infertility or developing new contraceptives.
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Gonadotropinas , Células de la Granulosa , Ratones Noqueados , Proteínas Serina-Treonina Quinasas , Animales , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Femenino , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Ratones , Gonadotropinas/metabolismo , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Aromatasa/genética , Aromatasa/metabolismo , Fertilidad/genética , Fertilidad/efectos de los fármacos , Estradiol/farmacologíaRESUMEN
During the development of animal organs, various adverse stimuli or toxic environments can induce oxidative stress and delay ovarian development. Paeoniflorin (PF), the main active ingredient of the traditional Chinese herb Paeonia lactiflora Pall., has protective effects on various diseases by preventing oxidative stress. However, the mechanism by which PF attenuates oxidative damage in mouse ovaries remains unclear. We evaluated the protective effects of PF on ovaries in an H2O2-induced mouse oxidative stress model. The H2O2-induced mouse ovarian oxidative stress model was used to explore the protective effect of PF on ovarian development. Histology and follicular development were observed. We then detected related indicators of cell apoptosis, oxidative stress, and autophagy in mouse ovaries. We found that PF inhibited H2O2-induced ovarian cell apoptosis and ferroptosis and promoted granulosa cell proliferation. PF prevented oxidative stress by increasing nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression levels. In addition, the autophagic flux of ovarian cells was activated and was accompanied by increased lysosomal biogenesis. Moreover, PF-mediated autophagy was involved in clearing mitochondria damaged by H2O2. Importantly, PF administration significantly increased the number of primordial follicles, primary follicles, secondary follicles, and antral follicles. PF administration improved ovarian sizes compared with the H2O2 group. The present study suggested that PF administration reversed H2O2-induced ovarian developmental delay and promoted follicle development. PF-activated mitophagy is crucial for preventing oxidative stress and improving mitochondrial quality.
Asunto(s)
Glucósidos , Peróxido de Hidrógeno , Mitofagia , Ovario , Estrés Oxidativo , Animales , Femenino , Estrés Oxidativo/efectos de los fármacos , Glucósidos/farmacología , Ratones , Ovario/efectos de los fármacos , Ovario/metabolismo , Mitofagia/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Monoterpenos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Proliferación Celular/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismoRESUMEN
Quercetin has been shown to mitigate the cytotoxic effects of heavy metals. While copper is an essential trace element for bodily functions, excessive intake has been linked to impaired female reproductive function. Transcriptome analysis was employed to identify genes that are differentially expressed in response to high copper and were validated through qRT-PCR and western blotting. ATP content and Tunel were used to identify the damage of mitochondrial and cell apoptosis. PPI analysis revealed that MKI67, TOPII, ASPM, CASP3, PLK1, and TTK are central proteins within the network. Additionally, exposure to elevated levels of copper resulted in the dysregulation of 86 genes associated with mitochondria. Conversely, treatment with quercetin (QUE) in combination with high copper led to the normalization of 42 mitochondria-related genes previously affected by high copper levels. Furthermore, CuSO4 decreases ATP content and induces cell apoptosis, which can be reversed by QUE. Results suggest that elevated copper levels could lead to oxidative stress and apoptosis by inducing mitochondrial damage, while QUE has the potential to mitigate these effects, ultimately safeguarding granulosa cells and halting the progression of cell death. This study provides novel insights into the molecular pathways involved in female reproductive toxicity caused by excessive copper exposure.
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Apoptosis , Cobre , Perfilación de la Expresión Génica , Quercetina , Femenino , Quercetina/farmacología , Cobre/toxicidad , Animales , Apoptosis/efectos de los fármacos , Reproducción/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Transcriptoma/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
Insulin resistance (IR) is one of the major complications of polycystic ovary syndrome (PCOS). This study aimed to investigate the effects and the molecular regulatory mechanism by which Dendrobium nobile-derived polysaccharides (DNP) improve IR in rats with letrozole and high-fat-diet induced PCOS. In vivo, DNP (200 mg/kg/d) administration not only reduced body weight, blood glucose, and insulin levels in PCOS rats, but also improve the disrupted estrous cycle. In addition, DNP treatment reduced atretic and cystic follicles and enhanced granulosa cell layer thickness, thereby restoring follicle development. In vitro, DNP treatment (100 µM) increased lactate levels and decreased pyruvate levels in insulin-treated (8 µg/mL) KGN cells. Additionally, DNP also decreased the expression of IGF1 and increased that of IGF1R, SIRT2, LDHA, PKM2 and HK2 both in vivo and in vitro. Also, SIRT2 expression was specifically inhibited by AGK2, while DNP significantly improved IR and glycolysis by reversing the effect of AGK2 treatment on lactate and pyruvate production, upregulating the expression levels of IGF1R, LDHA, HK2, and PKM2 and downregulating the expression level of IGF1. The results indicate that DNP can effectively improve IR and restore glycolytic pathway by activating SIRT2, which may provide a potential therapeutic approach for PCOS patients.
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Dendrobium , Glucólisis , Células de la Granulosa , Resistencia a la Insulina , Síndrome del Ovario Poliquístico , Polisacáridos , Sirtuina 2 , Síndrome del Ovario Poliquístico/metabolismo , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Femenino , Animales , Polisacáridos/farmacología , Glucólisis/efectos de los fármacos , Células de la Granulosa/metabolismo , Células de la Granulosa/efectos de los fármacos , Ratas , Dendrobium/química , Sirtuina 2/metabolismo , Sirtuina 2/genética , Humanos , Insulina/metabolismo , Ratas Sprague-DawleyRESUMEN
Chemotherapy-induced premature ovarian insufficiency (CIPOI) triggers gonadotoxicity in women undergoing cancer treatment, leading to loss of ovarian reserves and subfertility, with no effective therapies available. In our study, fecal microbiota transplantation in a cisplatin-induced POI mouse model reveals that a dysbiotic gut microbiome negatively impacts ovarian health in CIPOI. Multi-omics analyses show a significant decrease in Limosilactobacillus reuteri and its catabolite, ß-resorcylic acid , in the CIPOI group in comparison to healthy controls. Supplementation with L. reuteri or ß-RA mitigates cisplatin-induced hormonal disruptions, morphological damages, and reductions in follicular reserve. Most importantly, ß-RA pre-treatment effectively preserves oocyte function, embryonic development, and fetus health, thereby protecting against chemotherapy-induced subfertility. Our results provide evidence that ß-RA suppresses the nuclear accumulation of sex-determining region Y-box 7, which in turn reduces Bcl-2-associated X activation and inhibits granulosa cell apoptosis. These findings highlight the therapeutic potential of targeting the gut-ovary axis for fertility preservation in CIPOI.
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Cisplatino , Limosilactobacillus reuteri , Ovario , Insuficiencia Ovárica Primaria , Femenino , Animales , Cisplatino/efectos adversos , Cisplatino/toxicidad , Ratones , Insuficiencia Ovárica Primaria/inducido químicamente , Insuficiencia Ovárica Primaria/patología , Ovario/efectos de los fármacos , Ovario/patología , Ovario/metabolismo , Microbioma Gastrointestinal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Trasplante de Microbiota Fecal , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Ratones Endogámicos C57BL , Antineoplásicos/toxicidad , Antineoplásicos/efectos adversos , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Modelos Animales de Enfermedad , InfertilidadRESUMEN
BACKGROUND: At present, a number of clinical trials have been carried out on GLP-1 receptor agonist liraglutide in the treatment of polycystic ovary syndrome (PCOS). However, the effect of liraglutide on follicle development and its specific mechanism are still unclear. METHODS: RNA sequencing was used to explore the molecular characteristics of granulosa cells from patients with PCOS treated with liraglutide. The levels of C-X-C motif chemokine ligand 10 (CXCL10) in follicular fluid were detected by ELISA, the expression levels of ovulation related genes and inflammatory factor genes in follicles and granulosa cells were detected by qPCR and the protein levels of connexin 43 (Cx43), Janus Kinase 2 (JAK2) and phosphorylated JAK2 were detected by Western blot. The mouse ovarian follicles culture system in vitro was used to detect the status of follicle development and ovulation. RESULTS: In the present study, we found that liraglutide inhibited the secretion of inflammatory factors in PCOS granulosa cells, among which CXCL10 was the most significant. In addition, CXCL10 was significantly higher in granulosa cells and follicular fluid in PCOS patients than in non-PCOS patients. We applied in vitro follicle culture and other techniques to carry out the mechanism exploration which revealed that CXCL10 disrupted the homeostasis of gap junction protein alpha 1 (GJA1) between oocyte and granulosa cells before physiological ovulation, thus inhibiting follicular development and ovulation. Liraglutide inhibited CXCL10 secretion in PCOS granulosa cells by inhibiting the JAK signaling pathway and can improved dehydroepiandrosterone (DHEA)-induced follicle development disorders, which is reversed by CXCL10 supplementation. CONCLUSIONS: The present study suggests that liraglutide inhibits CXCL10 secretion in granulosa cells through JAK signaling pathway, thereby improving the homeostasis of GJA1 between oocyte and granulosa cells before physiological ovulation and ultimately improving the follicular development and ovulation of PCOS, which provides more supportive evidence for the clinical application of liraglutide in the treatment of ovulatory disorders in PCOS. TRIAL REGISTRATION: Not applicable.
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Quimiocina CXCL10 , Células de la Granulosa , Liraglutida , Folículo Ovárico , Síndrome del Ovario Poliquístico , Síndrome del Ovario Poliquístico/metabolismo , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Femenino , Liraglutida/farmacología , Liraglutida/uso terapéutico , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/genética , Humanos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Animales , Ratones , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Adulto , Ovulación/efectos de los fármacos , Líquido Folicular/metabolismo , Células Cultivadas , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéuticoRESUMEN
BACKGROUND: Zishen Qingre Lishi Huayu recipe (ZQLHR) has shown significant therapeutic effects in treating sex hormone levels and follicular developmental disorders in patients with polycystic ovary syndrome (PCOS). However, little is known about the potential mechanisms of its treatment. METHODS: Dehydroepiandrosterone and a high-fat diet induced the PCOS model rat. The serum of rats was collected to detect the levels of sex hormones and inflammatory cytokines by enzyme-linked immunosorbent assay, and the ovaries were collected for ovarian histopathology and qPCR assay to detect the levels of inflammatory cytokines in ovarian tissues. Granulosa cells (GCs) were collected for western blot assay to detect of IL-1ß, IL-6R, and LOX protein expression levels. RESULTS: ZQLHR could reduce body weight, regulate estrous cycles, and improve serum sex hormone levels, follicular development, and insulin resistance (IR) in PCOS model rats. In addition, ZQLHR treatment improved the levels of inflammatory cytokines in serum and ovary, and regulated the protein expression of IL-6R, IL-1ß, and LOX in GCs of PCOS model rats. The results showed that the HOMA-IR index increased with the increasing levels of IL-6, IL-1ß, and CRP, and decreased with the increased IL-10. CONCLUSION: This study reveals that the treatment of endocrine disorders and ovulation disorders in PCOS with ZQLHR may be closely related to the improvement of systemic and ovarian inflammation in PCOS patients, as well as the inhibition of IL-6R, IL-1ß, and LOX expression in GCs, which reemphasizes the role of reducing chronic inflammatory states in the treatment of PCOS. Moreover, this study reemphasizes the correlation between multiple inflammatory mediators and IR.
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Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos , Inflamación , Ovario , Síndrome del Ovario Poliquístico , Animales , Femenino , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Síndrome del Ovario Poliquístico/metabolismo , Ratas , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Inflamación/tratamiento farmacológico , Ovario/patología , Ovario/efectos de los fármacos , Ovario/metabolismo , Ratas Sprague-Dawley , Citocinas/metabolismo , Humanos , Dieta Alta en Grasa , Células de la Granulosa/metabolismo , Células de la Granulosa/efectos de los fármacos , Resistencia a la Insulina , Interleucina-1beta/metabolismo , Interleucina-1beta/sangreRESUMEN
The pathologic mechanism of polycystic ovary syndrome (PCOS) is related to increased autophagy of granulosa cells. Both berberine and metformin have been shown to improve PCOS, but whether the combination of berberine and metformin can better improve PCOS by inhibiting autophagy remains unclear. PCOS models were constructed by injecting dehydroepiandrosterone into rats, and berberine, metformin or berberine combined with metformin was administered to rats after modeling. Rats' body weight and ovarian weight were measured before and after modeling. Histopathological examination of ovarian tissue and estrous cycle analysis of rats were performed. Insulin resistance, hormone levels, oxidative stress, and lipid metabolism in PCOS rats were assessed. Expression of the AMPK/AKT/mTOR pathway and autophagy-related proteins was analyzed by Western blot assays. Granulosa cells were isolated from rat ovarian tissue and identified by immunofluorescence staining followed by transmission electron microscopy analysis. Berberine combined with metformin reduced the body weight and ovarian weight of PCOS rats, increased the number of primordial and primary follicles, decreased the number of secondary and atretic follicles, normalized the estrous cycle, and improved insulin resistance, androgen biosynthesis, oxidative stress and lipid metabolism disorders, and increased estrogen production. In addition, berberine combined with metformin reduced the number of autophagosomes in granulosa cells, which may be related to AMPK/AKT/mTOR pathway activation, decreased Beclin1 and LC3II/LC3I levels, and increased p62 expression. Berberine combined with metformin could inhibit autophagy by activating the AMPK/AKT/mTOR pathway in PCOS, indicating that berberine combined with metformin is a potential treatment strategy for PCOS.
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Autofagia , Berberina , Metformina , Síndrome del Ovario Poliquístico , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Serina-Treonina Quinasas TOR , Femenino , Animales , Metformina/farmacología , Síndrome del Ovario Poliquístico/metabolismo , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Síndrome del Ovario Poliquístico/patología , Autofagia/efectos de los fármacos , Berberina/farmacología , Ratas , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Ratas Sprague-Dawley , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Células de la Granulosa/patología , Resistencia a la Insulina , Ovario/efectos de los fármacos , Ovario/metabolismo , Ovario/patología , Quimioterapia Combinada , Estrés Oxidativo/efectos de los fármacosRESUMEN
BACKGROUND: Nanoplastics (NPs) are emerging pollutants that pose risks to living organisms. Recent findings have unveiled the reproductive harm caused by polystyrene nanoparticles (PS-NPs) in female animals, yet the intricate mechanism remains incompletely understood. Under this research, we investigated whether sustained exposure to PS-NPs at certain concentrations in vivo can enter oocytes through the zona pellucida or through other routes that affect female reproduction. RESULTS: We show that PS-NPs disrupted ovarian functions and decreased oocyte quality, which may be a contributing factor to lower female fertility in mice. RNA sequencing of mouse ovaries illustrated that the PI3K-AKT signaling pathway emerged as the predominant environmental information processing pathway responding to PS-NPs. Western blotting results of ovaries in vivo and cells in vitro showed that PS-NPs deactivated PI3K-AKT signaling pathway by down-regulating the expression of PI3K and reducing AKT phosphorylation at the protein level, PI3K-AKT signaling pathway which was accompanied by the activation of autophagy and apoptosis and the disruption of steroidogenesis in granulosa cells. Since PS-NPs penetrate granulosa cells but not oocytes, we examined whether PS-NPs indirectly affect oocyte quality through granulosa cells using a granulosa cell-oocyte coculture system. Preincubation of granulosa cells with PS-NPs causes granulosa cell dysfunction, resulting in a decrease in the quality of the cocultured oocytes that can be reversed by the addition of 17ß-estradiol. CONCLUSIONS: This study provides findings on how PS-NPs impact ovarian function and include transcriptome sequencing analysis of ovarian tissue. The study demonstrates that PS-NPs impair oocyte quality by altering the functioning of ovarian granulosa cells. Therefore, it is necessary to focus on the research on the effects of PS-NPs on female reproduction and the related methods that may mitigate their toxicity.
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Células de la Granulosa , Nanopartículas , Oocitos , Poliestirenos , Transducción de Señal , Animales , Femenino , Ratones , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Fertilidad/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Nanopartículas/toxicidad , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Poliestirenos/toxicidad , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
3-nitropropanoic acid is a potent oxidative stress inducer that is conventionally regarded as a regulator of follicular atresia by regulating granulosa cells (GCs) death through the apoptosis pathway. There has been no research investigating the impact of copper metal overload induced Cuproptosis in ovarian GCs as a factor contributing to hindered follicular development.To elucidate whether 3-NP-induced oxidative stress plays a contributory role in promoting Cuproptosis, and discuss the role of Cuproptosis in the development of ovarian follicles.We conducted an analysis of cuproptosis occurrence in murine GCs and C57BL/6 J mice under the influence of 3-NP and 3-NP with added exogenous copper.The results revealed that 3-NP serving as a robust facilitator of exogenous copper uptake by upregulating the expression of copper transporter 1 (CTR1). In turn, culminated in the accumulation of intracellular copper within mouse granulosa cells (mGCs). Furthermore, 3-NP promoted mitochondrial permeability transition pore opening and concurrently reduced the stability of lipoic acid proteins. These actions collectively induced the oligomerization of Dihydrolipoamide S-Acetyltransferase (DLAT), ultimately leading to cuproptosis in GCs and consequent follicular atresia. Heavy metal copper and fungal decomposition product 3-NP, induce ovarian atresia via cuproptosis, modulating the reproductive performance of female animals.
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Cobre , Atresia Folicular , Células de la Granulosa , Ratones Endogámicos C57BL , Animales , Femenino , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Atresia Folicular/efectos de los fármacos , Cobre/toxicidad , Transportador de Cobre 1/metabolismo , Ratones , Estrés Oxidativo/efectos de los fármacos , Muerte Celular/efectos de los fármacosRESUMEN
BACKGROUND: Secreted frizzled-related proteins (SFRPs) comprise a family of WNT signaling antagonists whose roles in the ovary are poorly understood. Sfrp4-null mice were previously found to be hyperfertile due to an enhanced granulosa cell response to gonadotropins, leading to decreased antral follicle atresia and enhanced ovulation rates. The present study aimed to elucidate the mechanisms whereby SFRP4 antagonizes FSH action. METHODS: Primary cultures of granulosa cells from wild-type mice were treated with FSH and/or SFRP4, and effects of treatment on gene expression were evaluated by RT-qPCR and RNAseq. Bioinformatic analyses were conducted to analyse the effects of SFRP4 on the transcriptome, and compare them to those of FSH or a constitutively active mutant of FOXO1. Additional granulosa cell cultures from wild-type or Sfrp4-null mice, some pretreated with pharmacologic inhibitors of specific signaling effectors, were used to examine the effects of FSH and/or SFRP4 on signaling pathways, autophagy and apoptosis by western blotting and TUNEL. RESULTS: Treatment of cultured granulosa cells with recombinant SFRP4 was found to decrease basal and FSH-stimulated mRNA levels of FSH target genes. Unexpectedly, this effect was found to occur neither via a canonical (CTNNB1-dependent) nor non-canonical WNT signaling mechanism, but was found to be GSK3ß-dependent. Rather, SFRP4 was found to antognize AKT activity via a mechanism involving AMPK. This lead to the hypophosphorylation of FOXO1 and a decrease in the expression of a portion of the FSH and FOXO1 transcriptomes. Conversely, FSH-stimulated AMPK, AKT and FOXO1 phosphorylation levels were found to be increased in the granulosa cells of Sfrp4-null mice relative to wild-type controls. SFRP4 treatement of granulosa cells also induced autophagy by signaling via AKT-mTORC1-ULK1, as well as apoptosis. CONCLUSIONS: This study identifies a novel GSK3ß-AMPK-AKT signaling mechanism through which SFPR4 antagonizes FSH action, and further identifies SFRP4 as a novel regulator of granulosa cell autophagy. These findings provide a mechanistic basis for the phenotypic changes previously observed in Sfrp4-null mice, and broaden our understanding of the physiological roles of WNT signaling processes in the ovary.
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Autofagia , Hormona Folículo Estimulante , Células de la Granulosa , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Animales , Células de la Granulosa/metabolismo , Células de la Granulosa/efectos de los fármacos , Femenino , Proteínas Proto-Oncogénicas c-akt/metabolismo , Autofagia/efectos de los fármacos , Hormona Folículo Estimulante/farmacología , Hormona Folículo Estimulante/metabolismo , Ratones , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Ratones Endogámicos C57BL , Células Cultivadas , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Ratones NoqueadosRESUMEN
OBJECTIVE: The main purpose of this study was to elucidate the anti-apoptotic effects of curculigoside (CUR) on ovarian granulosa cells (GCs) in a mouse model of cyclophosphamide (CTX)-induced premature ovarian failure (POF). METHOD: Intraperitoneal injection of CTX (100 mg/kg body weight) induced POF in mice. Thirty-six female mice were divided into six groups: blank group; POF model group; low-dose CUR group; medium-dose CUR group; high-dose CUR group; and estradiol benzoate group. Mice were orally administered for 28 consecutive days. Twenty-four hours after the completion of treatment, mice were weighed and euthanized, and blood was collected from the eyeball under anesthesia. The ovaries were surgically separated and weighed, and the ovarian index was calculated. Hematoxylin-eosin (HE) staining was used to observe follicular development and corpus luteum morphology in the ovaries. Serum levels of follicle stimulating hormone (FSH), anti-Müllerian hormone (AMH) and estradiol (E2) were measured. Superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) content and malondialdehyde (MDA) levels in ovarian tissue were determined. The GC apoptosis level was measured. Western blotting was used to detect protein expression levels of Beclin-1, LC3, P62, AKT, p-AKT, mTOR and p-mTOR in the ovaries. RESULTS: The results showed that CUR can improve body weight and ovarian index; promote follicular development and reduce follicular atresia; improve FSH, AMH and E2 levels; downregulate MDA levels and restore antioxidant enzyme activity; inhibit the autophagy level; activate the AKT/mTOR signaling pathway; and alleviate GC apoptosis. CONCLUSION: CUR improves POF by activating the AKT/mTOR signaling pathway, inhibiting autophagy and alleviating GC apoptosis.
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Apoptosis , Ciclofosfamida , Modelos Animales de Enfermedad , Glucósidos , Células de la Granulosa , Insuficiencia Ovárica Primaria , Animales , Femenino , Ciclofosfamida/efectos adversos , Insuficiencia Ovárica Primaria/inducido químicamente , Insuficiencia Ovárica Primaria/tratamiento farmacológico , Ratones , Glucósidos/farmacología , Apoptosis/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Estradiol/sangre , Ovario/efectos de los fármacos , Ovario/patología , Hormona Folículo Estimulante/sangre , Serina-Treonina Quinasas TOR/metabolismo , Transducción de Señal/efectos de los fármacos , Malondialdehído/metabolismo , Hormona Antimülleriana/sangre , BenzoatosRESUMEN
Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control antral follicular growth by regulating several processes, such as the synthesis of hormones and signaling molecules, proliferation, survival, apoptosis, luteinization, and ovulation. To exert these effects, gonadotropins bind to their respective Gs protein-coupled receptors, activating the protein kinase A (PKA) pathway or recruiting Gq proteins to activate protein kinase C (PKC) signaling. Although the action mechanism of FSH and LH is clear, recently, it has been shown that both gonadotropins promote the synthesis of sphingosine-1-phosphate (S1P) in granulosa and theca cells through the activation of sphingosine kinase 1. Moreover, the inhibition of SPHKs reduces S1P synthesis, cell viability, and the proliferation of follicular cells in response to gonadotropins, and the addition of S1P to the culture medium increases the proliferation of granulosa and theca cells without apparent effects on sexual steroid synthesis. Therefore, we consider that S1P is a crucial signaling molecule that complements the canonical gonadotropin pathway to promote the proliferation and viability of granulosa and theca cells.
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Gonadotropinas , Lisofosfolípidos , Folículo Ovárico , Esfingosina , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacología , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Femenino , Animales , Humanos , Gonadotropinas/metabolismo , Folículo Ovárico/metabolismo , Folículo Ovárico/efectos de los fármacos , Hormona Luteinizante/metabolismo , Hormona Folículo Estimulante/metabolismo , Hormona Folículo Estimulante/farmacología , Transducción de Señal/efectos de los fármacos , Células de la Granulosa/metabolismo , Células de la Granulosa/efectos de los fármacosRESUMEN
T-2 toxin, a mycotoxin found in foods and feeds, poses a threat to female reproductive health in both humans and animals. LncRNA CUFF.253988.1 (CUFF.253988.1), highly expressed in pigs, has an undisclosed regulatory role. This study aimed to establish a model of T-2 toxin-induced ovarian injury in sows, both in vivo and in vitro, and to explore the regulatory role and potential mechanisms of CUFF.253988.1. The results showed that feeding T-2 toxin-contaminated feed (1â¯mg/kg) induced ovarian follicle atresia and mitochondrial structural damage, accompanied by a significant upregulation of CUFF.253988.1 expression in the ovaries. Additionally, T-2 toxin inhibited the SIRT3/PGC1-α pathway associated with mitochondrial function. Moreover, T-2 toxin induced cell apoptosis by upregulating the expression of Cyt c, Bax, cleaved-caspase-9, and cleaved-caspase-3 proteins. In T-2 toxin-induced injury to the ovarian granulosa AVG-16 cells at concentrations of 10, 40 and 160â¯nM, not only were the previously mentioned effects observed, but also a decrease in mitochondrial membrane potential, ATP content, and an elevation in ROS levels. However, downregulating CUFF.253988.1 reversed T-2 toxin's inhibition of the SIRT3/PGC1-α pathway, alleviating mitochondrial dysfunction and reducing cell apoptosis. Notably, this may be attributed to the inhibition of T-2 toxin-induced enrichment of CUFF.253988.1 in mitochondria. In conclusion, CUFF.253988.1 plays a pivotal role in T-2 toxin-induced ovarian damage, operating through the inhibition of the SIRT3/PGC1-α pathway and promotion of cell apoptosis.
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Apoptosis , Ovario , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , ARN Largo no Codificante , Sirtuina 3 , Toxina T-2 , Animales , Femenino , Apoptosis/efectos de los fármacos , Toxina T-2/toxicidad , Sirtuina 3/genética , Sirtuina 3/metabolismo , Porcinos , ARN Largo no Codificante/genética , Ovario/efectos de los fármacos , Ovario/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Células de la Granulosa/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Di(2-ethylhexyl) phthalate (DEHP) is a widely recognized environmental endocrine disruptor that potentially impacts female reproductive function, although the specific mechanisms leading to such impairment remain unclear. A growing body of research has revealed that the endoplasmic reticulum and mitochondrial function significantly influence oocyte quality. The structure of mitochondria-associated endoplasmic reticulum membranes (MAMs) is crucial for facilitating the exchange of Ca2+, lipids, and metabolites. This study aimed to investigate the alterations in the composition and function of MAMs after DEHP exposure and to elucidate the underlying mechanisms of ovarian toxicity. The female mice were exposed to DEHP at doses of 5 and 500â¯mg/kg/day for one month. The results revealed that DEHP exposure led to reduced serum anti-Müllerian hormone levels and increased atretic follicles in mice. DEHP induced endoplasmic reticulum stress and disrupted calcium homeostasis in oocytes. Furthermore, DEHP impaired the mitochondrial function of oocytes and reduced their membrane potential, and promoting apoptosis. Similar results were observed in human granulosa cells after exposure to mono-(2-ethylhexyl) phthalate (MEHP, metabolites of DEHP) in vitro. Proteomic analysis and transmission electron microscopy revealed modifications in the functional proteins and structure of the MAMs, and the suppression of oxidative phosphorylation pathways. The findings of this investigation provide a new perspective on the mechanism underlying the reproductive toxicity of DEHP in females.