RESUMEN
Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation. Here, we unveil mouse ANKRD31 as a lynchpin governing multiple aspects of DSB formation. Spermatocytes lacking ANKRD31 have altered DSB locations and fail to target DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. They also have delayed and/or fewer recombination sites but, paradoxically, more DSBs, suggesting DSB dysregulation. Unrepaired DSBs and pairing failures-stochastic on autosomes, nearly absolute on X and Y-cause meiotic arrest and sterility in males. Ankrd31-deficient females have reduced oocyte reserves. A crystal structure defines a pleckstrin homology (PH) domain in REC114 and its direct intermolecular contacts with ANKRD31. In vivo, ANKRD31 stabilizes REC114 association with the PAR and elsewhere. Our findings inform a model in which ANKRD31 is a scaffold anchoring REC114 and other factors to specific genomic locations, thereby regulating DSB formation.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Recombinación Homóloga/genética , Meiosis/genética , Recombinasas/química , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Emparejamiento Cromosómico , Segregación Cromosómica/genética , Cromosomas , Cristalografía por Rayos X , Roturas del ADN de Doble Cadena , Femenino , Masculino , Ratones , Conformación Proteica , Recombinasas/genética , Espermatocitos/química , Espermatocitos/metabolismoRESUMEN
In meiosis, homologous chromosome synapsis is mediated by a supramolecular protein structure, the synaptonemal complex (SC), that assembles between homologous chromosome axes. The mammalian SC comprises at least eight largely coiled-coil proteins that interact and self-assemble to generate a long, zipper-like structure that holds homologous chromosomes in close proximity and promotes the formation of genetic crossovers and accurate meiotic chromosome segregation. In recent years, numerous mutations in human SC genes have been associated with different types of male and female infertility. Here, we integrate structural information on the human SC with mouse and human genetics to describe the molecular mechanisms by which SC mutations can result in human infertility. We outline certain themes in which different SC proteins are susceptible to different types of disease mutation and how genetic variants with seemingly minor effects on SC proteins may act as dominant-negative mutations in which the heterozygous state is pathogenic.
Asunto(s)
Infertilidad , Complejo Sinaptonémico , Masculino , Femenino , Humanos , Ratones , Animales , Complejo Sinaptonémico/genética , Emparejamiento Cromosómico , Meiosis/genética , Infertilidad/genética , Mutación , Mamíferos/genéticaRESUMEN
Recent large-scale mRNA sequencing has shown that introns are retained in 5-10% of mRNA, and these events are named intron retention (IR). IR has been recognized as a key mechanism in the regulation of gene expression. However, the role of this mechanism in female reproduction in mammals remains unclear. RNA terminal phosphate cyclase B (RTCB) is a RNA ligase; we found that RTCB conditional knockout mice have premature ovarian failure and that RTCB plays a crucial role in follicular development. RTCB regulated the splicing of transcripts related to DNA methylation and DNA damage repair. In addition, it regulated the resumption of oocyte meiosis by affecting CDK1 activation. Moreover, the loss of RTCB suppressed zygotic genome activation (ZGA) and decreased translation at the global level. In addition, Rtcb deletion resulted in the accumulation of maternal mRNAs containing unspliced introns and in a decline in the overall level of transcripts. As a result, the Rtcb-/- females were sterile. Our study highlights the important role of RTCB-regulated noncanonical alternative splicing in female reproduction.
Asunto(s)
Empalme Alternativo , Aminoacil-ARNt Sintetasas/metabolismo , Fosfatos , Empalme Alternativo/genética , Animales , Femenino , Ligasas/genética , Mamíferos/genética , Ratones , Oocitos , Empalme del ARN , ARN Mensajero/genéticaRESUMEN
STUDY QUESTION: Does platelet-rich plasma (PRP) intraovarian injection increase the number of retrieved oocytes in successive ovarian punctions among patients with poor ovarian reserve (POR)? SUMMARY ANSWER: The injection of PRP increases the number of retrieved oocytes without increasing the quality of developed blastocysts. WHAT IS KNOWN ALREADY: Management of women with reduced ovarian response to stimulation is one of the significant challenges in reproductive medicine. Recently, PRP treatment has been proposed as an adjunct in assisted reproduction technology, with controversial results. STUDY DESIGN, SIZE, DURATION: This placebo-controlled, double-blind, randomized trial included 60 patients with POR stratified according to the POSEIDON classification groups 3 and 4. It was conducted to explore the efficacy and safety of intraovarian PRP injection. Patients were proposed to undergo three consecutive ovarian stimulations to accumulate oocytes and were randomized to receive either PRP or placebo during their first oocyte retrieval. Randomization was performed using computer-generated randomization codes. Double blinding was ensured so that neither the participant nor the investigators knew of the treatment allotted. All patients underwent three ovarian stimulations and egg retrieval procedures. ICSI was performed after a third ovarian puncture. The primary endpoint was the number of mature oocytes retrieved after PRP or placebo injection in successive ovarian punctures. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sixty women (30-42 years) fulfilling inclusion criteria were randomized in equal proportions to the treatment or control groups. MAIN RESULTS AND THE ROLE OF CHANCE: The baseline demographic and clinical characteristics [age, BMI, anti-Müllerian hormone (AMH) levels] were comparable between the groups. Regarding the primary endpoint, the cumulative number (mean ± SEM) of retrieved mature oocytes was slightly higher in the treatment group: 10.45 ± 0.41 versus 8.91 ± 0.39 in the control group, respectively (95% CI of the difference 0.42-2.66; P = 0,008). The number of mature oocytes obtained among all patients increased in successive egg retrievals: 2.61 ± 0.33 (mean ± SEM) in punction 1 (P1), 3.85 ± 0.42 in P2, and 4.73 ± 0.44 in P3. However, the increase was higher among patients receiving the assessed PRP treatment. In P2, the number of retrieved mature oocytes was 4.18 ± 0.58 versus 3.27 ± 0.61 in controls (95% CI of the difference: -0.30 to 2.12; P = 0.138) and in P3, 5.27 ± 0.73 versus 4.15 ± 0.45 (95% CI of the difference: 0.12-2.12; P = 0.029). The mean ± SEM number of developed and biopsied blastocysts was 2.43 ± 0.60 in the control group and 1.90 ± 0.32 in the treatment group, respectively (P = 0.449). The mean number of euploid blastocysts was 0.81 ± 0.24 and 0.81 ± 0.25 in the control and treatment groups, respectively (P = 1.000). The percentages of patients with euploid blastocysts were 53.33% (16 out of 30) and 43.33% (13 out of 30) for patients in the control and treatment groups, respectively (Fisher's exact test P = 0.606). The overall pregnancy rate per ITT was 43% (26 out of 60 patients). However, the percentage of clinical pregnancies was higher in the control group (18 out of 30, 60%) than in the treatment group (8 out of 30, 27%) (P = 0.018). There was also a trend toward poorer outcomes in the treatment group when considering full-term pregnancies (P = 0.170). There were no differences between control and treatment groups regarding type of delivery, and sex of newborns. LIMITATIONS, REASONS FOR CAUTION: The mechanism of the potential beneficial effect of PRP injection on the number of retrieved oocytes is unknown. Either delivered platelet factors or a mechanical effect could be implicated. Further studies will be needed to confirm or refute the data presented in this trial and to specify the exact mechanism of action, if any, of PRP preparations. WIDER IMPLICATIONS OF THE FINDINGS: The increasing number of women with a poor response to ovarian stimulation supports the exploration of new areas of research to know the potential benefits of therapies capable of increasing the number of oocytes available for fertilization and improving the quality of developed blastocysts. An increase in the retrieved oocytes in both arms of the trial suggests that, beyond the release of growth factor from platelets, a mechanical effect can play a role. However, neither improvement in euploid blastocyst development nor pregnancy rates have been demonstrated. STUDY FUNDING/COMPETING INTEREST(S): This trial was supported by Basque Government and included in HAZITEK program, framed in the new Euskadi 2030 Science and Technology Plan (PCTI 2030). These aids are co-financed by the European Regional Development Fund (FEDER). The study funders had no role in the study design, implementation, analysis, manuscript preparation, or decision to submit this article for publication. No competing interests are declared by all the authors. TRIAL REGISTRATION NUMBER: Clinical Trial Number EudraCT 2020-000247-32. TRIAL REGISTRATION DATE: 3 November 2020. DATE OF FIRST PATIENT'S ENROLLMENT: 16 January 2021.
Asunto(s)
Fertilización In Vitro , Técnicas Reproductivas Asistidas , Recién Nacido , Embarazo , Humanos , Femenino , Fertilización In Vitro/métodos , Resultado del Tratamiento , Ovario , Índice de Embarazo , Inducción de la Ovulación/métodosRESUMEN
BACKGROUND AND AIM: Cyclophosphamide (CP) chemotherapy is a significant iatrogenic component of premature ovarian failure (POF). The aim of this work was to evaluate the potential protective effects of donepezil, a centrally acting acetylcholinesterase (AChE) inhibitor, on CP-induced POF in mice. METHODS: 40 female Swiss albino mice were split into 5 equal groups: group 1 (control), group 2 (CP-POF); induced by intraperitoneal injection of CP on 8th day of the experiment, and group (3-5); mice received oral donepezil daily (1, 2, or 4 mg/kg, respectively) 8 days before CP injection. Mice were euthanized after 24 h of CP injection, and blood samples were collected to assay serum anti-Mullerian hormone (AMH) levels. Ovarian tissues were dissected, and the right ovary was processed for further assays of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interlukin-6 (IL-6), nucleotide-binding domain-like receptor family, the Pyrin domain-containing 3 (NLRP3) inflammasome, and Toll-like receptor 4 (TLR-4), while the left one was processed for histopathological and immunohistochemical examination of nuclear factor-Kappa beta (NF-κB) and caspase-3. RESULTS: Donepezil, in a dose-dependent manner particularly (4 mg/kg), has an inhibitory action on NO (40 ± 2.85 vs. 28.20 ± 2.23, P < 0.001), proinflammatory cytokines (P < 0.001), the TLR-4/ NF-κB / NLRP3 inflammasome pathway (P < 0.001), and apoptosis (P < 0.001), with a significant elevation in the AMH levels (4.57 ± 1.08 vs. 8.57 ± 0.97, P < 0.001) versus CP-POF group. CONCLUSION: Donepezil may be a potential protective agent against CP-induced POF in mice, but further research is needed to fully understand its therapeutic function experimentally and clinically.
Asunto(s)
Inhibidores de la Colinesterasa , Ciclofosfamida , Citocinas , Donepezilo , FN-kappa B , Proteína con Dominio Pirina 3 de la Familia NLR , Insuficiencia Ovárica Primaria , Receptor Toll-Like 4 , Animales , Femenino , Donepezilo/farmacología , Ratones , Receptor Toll-Like 4/metabolismo , Ciclofosfamida/toxicidad , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Citocinas/metabolismo , Insuficiencia Ovárica Primaria/inducido químicamente , Insuficiencia Ovárica Primaria/prevención & control , Insuficiencia Ovárica Primaria/patología , Inhibidores de la Colinesterasa/farmacología , Ovario/efectos de los fármacos , Ovario/metabolismo , Ovario/patología , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Premature ovarian failure (POF) caused by cisplatin is a severe and intractable sequela for young women with cancer who received chemotherapy. Cisplatin causes the dysfunction of granulosa cells and mainly leads to but is not limited to its apoptosis and autophagy. Ferroptosis has been also reported to participate, while little is known about it. Our previous experiment has demonstrated that endometrial stem cells (EnSCs) can repair cisplatin-injured granulosa cells. However, it is still unclear whether EnSCs can play a repair role by acting on ferroptosis. METHODS: Western blotting and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were applied to detect the expression levels of ferroptosis-related genes. CCK-8 and 5-Ethynyl-2'-deoxyuridine (EdU) assays were used to evaluate cell viability. Transmission electron microscopy (TEM) was performed to detect ferroptosis in morphology. And the extent of ferroptosis was assessed by ROS, GPx, GSSG and MDA indicators. In vivo, ovarian morphology was presented by HE staining and the protein expression in ovarian tissue was detected by immunohistochemistry. RESULTS: Our results showed that ferroptosis could occur in cisplatin-injured granulosa cells. Ferroptosis inhibitor ferrostatin-1 (Fer-1) and EnSCs partly restored cell viability and mitigated the damage of cisplatin to granulosa cells by inhibiting ferroptosis. Moreover, the repair potential of EnSCs can be markedly blocked by ML385. CONCLUSION: Our study demonstrated that cisplatin could induce ferroptosis in granulosa cells, while EnSCs could inhibit ferroptosis and thus exert repair effects on the cisplatin-induced injury model both in vivo and in vitro. Meanwhile, Nrf2 was validated to participate in this regulatory process and played an essential role.
Asunto(s)
Cisplatino , Ferroptosis , Factor 2 Relacionado con NF-E2 , Femenino , Humanos , Cisplatino/farmacología , Cisplatino/uso terapéutico , Células de la Granulosa/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Células Madre/metabolismoRESUMEN
BACKGROUND: Ovarian damage and follicle loss are major side effects of chemotherapy in young female patients with cancer. However, effective strategies to prevent these injuries are still lacking. The purpose of this study was to verify low-intensity pulsed ultrasound (LIPUS) can reduce ovarian injury caused by chemotherapy and to explore its underlying mechanisms in mice model. METHODS: The mice were randomly divided into the Control group, Cisplatin group, and Cisplatin + LIPUS group. The Cisplatin group and Cisplatin + LIPUS group were intraperitoneally injected with cisplatin every other day for a total of 10 injections, and the Control group was injected with saline. On the second day of each injection, the Cisplatin + LIPUS group received irradiation, whereas the other two groups received sham irradiation. We used a variety of biotechnologies to detect the differences in follicle count, granulosa cell apoptosis, fibrosis, transcriptome level, oxidative damage, and inflammation in differently treated mice. RESULT: LIPUS was able to reduce primordial follicle pool depletion induced by cisplatin and inhibit the apoptosis of granulosa cells. Transcriptomic results confirmed that LIPUS can reduce ovarian tissue injury. We demonstrated that LIPUS can relieve ovarian fibrosis by inhibiting TGF-ß1/Smads pathway. Meanwhile, it can reduce the oxidative damage and reduced the mRNA levels of proinflammatory cytokines caused by chemotherapy. CONCLUSION: LIPUS can reduce the toxic effects of chemotherapy drugs on ovaries, inhibit ovarian fibrosis, reduce the inflammatory response, and redcue the oxidative damage, reduce follicle depletion and to maintain the number of follicle pools.
Asunto(s)
Antineoplásicos , Cisplatino , Ovario , Ondas Ultrasónicas , Animales , Femenino , Ratones , Cisplatino/efectos adversos , Ovario/efectos de los fármacos , Ovario/efectos de la radiación , Ovario/patología , Antineoplásicos/efectos adversos , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/efectos de la radiación , Terapia por Ultrasonido/métodosRESUMEN
Premature ovarian failure (POF) is a complication of ovarian dysfunction resulting from the depletion or dysfunction of primordial follicles (PFs) in the ovaries. However, residual follicles that have the potential to be activated are present in POF or aged women. Little is known about the mechanisms by which the remaining dormant PFs in POF patients are activated. Using mass spectrometry, we screened differentially generated peptides extracted from the ovarian cortical tissue biopsies of patients with or without POF, during which we identified PFAP1, a peptide that significantly promoted the activation of PFs in the ovaries of 3 dpp mice in vitro. PFAP1 reversed age-related fertility damage in vivo to a certain extent, promoted estrogen (E2) and anti-mullerian hormone (AMH) production (p < .05), and decreased the levels of follicle-stimulating hormone (FSH) (p < .05). In newborn mouse ovaries, PFAP1 could bind to the protein minichromosome maintenance protein 5 (MCM5) and inhibit its ubiquitination and degradation. In addition, PFAP1 promoted the proliferation of GCs, probably by regulating the function and production of MCM5. In conclusion, PFAP1 could promote the activation of PFs in the ovaries of newborn mice, partially restore the ovarian function of aged mice, and increase the proliferation of primary granulosa cells (GCs) by regulating the function of MCM5. PFAP1 is a promising novel peptide that may be developed into a new therapeutic agent for POF and other ovarian diseases.
Asunto(s)
Menopausia Prematura , Enfermedades del Ovario , Folículo Ovárico , Péptidos , Insuficiencia Ovárica Primaria , Animales , Femenino , Ratones , Hormona Antimülleriana , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Hormona Folículo Estimulante/metabolismo , Células de la Granulosa/metabolismo , Menopausia Prematura/metabolismo , Enfermedades del Ovario/tratamiento farmacológico , Enfermedades del Ovario/patología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Insuficiencia Ovárica Primaria/metabolismo , Péptidos/farmacologíaRESUMEN
RESEARCH QUESTION: Does type 1 diabetes mellitus (T1DM) affect reproductive health of female patients? What is the potential mechanism of reproductive dysfunction in female patients caused by T1DM? DESIGN: Preliminary assessment of serum levels of female hormones in women with or without T1DM. Then histological and immunological examinations were carried out on the pancreas, ovaries and uteri at different stages in non-obese diabetic (NOD) and Institute of Cancer Research (ICR) mice, as well as assessment of their fertility. A protein array was carried out to detect the changes in serum inflammatory cytokines. Furthermore, RNA-sequencing was used to identify the key abnormal genes/pathways in ovarian and uterine tissues of female NOD mice, which were further verified at the protein level. RESULTS: Testosterone levels were significantly increased (Pâ¯=â¯0.0036) in female mice with T1DM. Increasing age in female NOD mice was accompanied by obvious lymphocyte infiltration in the pancreatic islets. Moreover, the levels of serum inflammatory factors in NOD mice were sharply increased with increasing age. The fertility of female NOD mice declined markedly, and most were capable of conceiving only once. Furthermore, ovarian and uterine morphology and function were severely impaired in NOD female mice. Additionally, ovarian and uterine tissues revealed that the differentially expressed genes were primarily enriched in metabolism, cytokine-receptor interactions and chemokine signalling pathways. CONCLUSION: T1DM exerts a substantial impairment on female reproductive health, leading to diminished fertility, potentially associated with immune disorders and alterations in energy metabolism.
Asunto(s)
Diabetes Mellitus Tipo 1 , Islotes Pancreáticos , Humanos , Femenino , Animales , Ratones , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Ratones Endogámicos NOD , Páncreas/metabolismo , Páncreas/patología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Citocinas/metabolismo , Inflamación/metabolismoRESUMEN
Currently, no effective treatment exists for premature ovarian failure (POF). To obtain compounds with protective effects against POF, we aimed to design and synthesize a series of spiroheterocyclic protective agents with a focus on minimizing toxicity while enhancing their protective effect against cisplatin-induced POF. This was achieved through systematic modifications of Michael receptors and linkers within the molecular structure of 1,5-diphenylpenta-1,4-dien-3-one analogs. To assess the cytotoxicity and activity of these compounds, we constructed quantitative conformational relationship models using an artificial intelligence random forest algorithm, resulting in R2 values exceeding 0.87. Among these compounds, j2 exhibited optimal protective activity. It significantly increased the survival of cisplatin-injured ovarian granulosa KGN cells, improved post-injury cell morphology, reduced apoptosis, and enhanced cellular estradiol (E2) levels. Subsequent investigations revealed that j2 may exert its protective effect via a novel mechanism involving the activation of the SIRT1/AKT signal pathway. Furthermore, in cisplatin-injured POF in rats, j2 was effective in increasing body, ovarian, and uterine weights, elevating the number of follicles at all levels in the ovary, improving ovarian and uterine structures, and increasing serum E2 levels in rats with cisplatin-injured POF. In conclusion, this study introduces a promising compound j2 and a novel target SIRT1 with substantial protective activity against cisplatin-induced POF.
Asunto(s)
Cisplatino , Insuficiencia Ovárica Primaria , Sirtuina 1 , Femenino , Insuficiencia Ovárica Primaria/inducido químicamente , Insuficiencia Ovárica Primaria/tratamiento farmacológico , Insuficiencia Ovárica Primaria/patología , Insuficiencia Ovárica Primaria/metabolismo , Sirtuina 1/metabolismo , Sirtuina 1/antagonistas & inhibidores , Cisplatino/farmacología , Animales , Ratas , Humanos , Relación Estructura-Actividad , Regulación hacia Arriba/efectos de los fármacos , Ratas Sprague-Dawley , Estructura Molecular , Sustancias Protectoras/farmacología , Sustancias Protectoras/química , Sustancias Protectoras/síntesis química , Descubrimiento de Drogas , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Relación Dosis-Respuesta a Droga , Apoptosis/efectos de los fármacos , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/síntesis químicaRESUMEN
Long non-coding RNA (lncRNA) anomalies cause early ovarian failure. LncRNA nuclear enriched abundant transcript 1 (NEAT1) was down-regulated in premature ovarian failure (POF) mice and connected to the illness, however, the mechanism remained unclear. The levels of gene and protein were measured by using quantitative real-time polymerase chain reaction, Western blot, and immunofluorescence. Follicle stimulating hormone (FSH), estradiol (E2), and luteinizing hormone (LH) levels were determined using enzyme-linked immunosorbent assay (ELISA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and flow cytometry were used to determine cell viability and apoptosis. The interaction of NEAT1, miR-654, and stanniocalcin-2 (STC2) was verified by dual-luciferase reporter assay or RNA binding protein immunoprecipitation (RIP) assays. The results showed NEAT1 and STC2 down-regulated, while miR-654 up-regulated in POF mice. Overexpression of NEAT1 reduced apoptosis and autophagy in cyclophosphamide (CTX)-treated ovarian granulosa cells (OGCs), and Bax, cleaved-caspase3, LC3B, LC3II/LC3I ratio were decreased and Bcl-2 and p62 were raised. NEAT1 suppressed miR-654 expression by directly targeting miR-654. The inhibition of NEAT1 overexpression on apoptosis and autophagy in OGCs was reversed by miR-654 mimics. STC2 was a target gene of miR-654, and miR-654 inhibitor reduced the apoptosis and autophagy by regulating the STC2/MAPK axis. To sum up, NEAT1 reduced miR-654 expression and modulated the STC2/MAPK pathway to decrease apoptosis and autophagy in POF, indicating a potential therapeutic target.
Asunto(s)
Apoptosis , Autofagia , Células de la Granulosa , MicroARNs , ARN Largo no Codificante , Animales , Ratones , Apoptosis/genética , Autofagia/genética , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Transducción de Señal , Células de la Granulosa/metabolismo , Células de la Granulosa/patologíaRESUMEN
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.
Asunto(s)
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
BACKGROUND: Helicase for meiosis 1 (HFM1), a putative DNA helicase expressed in germ-line cells, has been reported to be closely associated with premature ovarian insufficiency (POI). However, the underlying molecular mechanism has not been clearly elucidated. The aim of this study was to investigate the function of HFM1 in the first meiotic prophase of mouse oocytes. RESULTS: The results suggested that the deficiency of HFM1 resulting in increased apoptosis and depletion of oocytes in mice, while the oocytes were arrested in the pachytene stage of the first meiotic prophase. In addition, impaired DNA double-strand break repair and disrupted synapsis were observed in the absence of HFM1. Further investigation revealed that knockout of HFM1 promoted ubiquitination and degradation of FUS protein mediated by FBXW11. Additionally, the depletion of HFM1 altered the intranuclear localization of FUS and regulated meiotic- and oocyte development-related genes in oocytes by modulating the expression of BRCA1. CONCLUSIONS: These findings elaborated that the critical role of HFM1 in orchestrating the regulation of DNA double-strand break repair and synapsis to ensure meiosis procession and primordial follicle formation. This study provided insights into the pathogenesis of POI and highlighted the importance of HFM1 in maintaining proper meiotic function in mouse oocytes.
Asunto(s)
Profase Meiótica I , Oocitos , Ubiquitinación , Animales , Femenino , Ratones , Apoptosis/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Meiosis/fisiología , Profase Meiótica I/fisiología , Ratones Noqueados , Oocitos/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genéticaRESUMEN
OBJECTIVE: To assess the impacts of Platelet-Rich Plasma(PRP) and Granulocyte Colony-Stimulating Factor(G-CSF) on a rat model with induced ovarian follicular damage caused by cyclophosphamide(Cy). MATERIALS AND METHODS: Forty-two Sprague-Dawley rats were randomly allocated into seven distinct groups as; Group 1(control): NaCl intraperitoneal (IP) injection was administered on days D1, D7, and D14. Group 2(Cy):Cy IP injection on D1 + NaCl IP injection on D7 and D14 were administered. Group 3(PRP): PRP IP injection on D1,D7 and D14 were administered. Group 4(Cy + PRP):Cy IP injection on D1 and PRP IP injection on D1, D7 and D14 were administered. Group 5(G-CSF): G-CSF IP injection on D1, D7 and D14 were administered. Group 6(Cy + G-CSF):Cy IP injection on D1+ G-CSF IP injection on D1, D7 and D14 were administered. Group 7(Cy + PRP + G-CSF):Cy IP injection on D1+ PRP IP injection on D1,D7 and D14+ G-CSF IP injection on D1,D7 and D14 were administered. Follicular number, histological scores of AMH and INSL3 stained follicles at different stages of follicular development, and serum Anti-Müllerian hormone(AMH) were evaluated. RESULTS: The primary, secondary, and antral follicle intensity scores for AMH-positive staining were most prominent in Groups 3 and 5. There was no significant difference between groups 4, 6 and 7 compared to group 1 in terms of follicule counts and AMH staining. The intensity scores of AMH-positive staining follicles were notably reduced in group 2 compared to groups 4, 6, and 7, with a significant difference (p < .01). Among the groups, group 2 exhibited the least intense antral follicle staining for INSL3, displaying a significant difference(p < .01) compared to the remaining groups. CONCLUSIONS: Autologous PRP and G-CSF might protect ovarian function in the face of ovarian damage caused by Cy-induced effects.
Asunto(s)
Hormonas Peptídicas , Plasma Rico en Plaquetas , Femenino , Ratas , Animales , Factor Estimulante de Colonias de Granulocitos/farmacología , Ratas Sprague-Dawley , Cloruro de Sodio , Inyecciones Intraperitoneales , Hormona AntimüllerianaRESUMEN
Premature ovarian insufficiency (POI) or premature ovarian failure (POF) is a multifactorial disorder occurring in reproductive-age women, characterized by elevated levels of follicle-stimulating hormone (FSH) and irregular or absent menstrual cycles, often accompanied by perimenopausal symptoms and infertility. While assisted reproductive technology can address the reproductive aspirations of some POI-affected women, it is hindered by issues such as exorbitant expenses, substantial risks, and poor rates of conception. Encouragingly, extensive research is exploring novel approaches to enhance fertility, particularly in the realm of stem cell therapy, showcasing both feasibility and significant potential. Human amniotic epithelial cells (hAECs) from discarded placental tissues are crucial in regenerative medicine for their pluripotency, low immunogenicity, non-tumorigenicity, accessibility, and minimal ethical concerns. Preclinical studies highlight the underlying mechanisms and therapeutic effects of hAECs in POI treatment, and current research is focusing on innovative interventions to augment hAECs' efficacy. However, despite these strides, overcoming application challenges is essential for successful clinical translation. This paper conducted a comprehensive analysis of the aforementioned issues, examining the prospects and challenges of hAECs in POI, with the aim of providing some insights for future research and clinical practice.
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Amnios , Células Epiteliales , Insuficiencia Ovárica Primaria , Humanos , Insuficiencia Ovárica Primaria/terapia , Femenino , Células Epiteliales/trasplante , Amnios/citología , Amnios/trasplanteRESUMEN
AIM: To investigate the DNA damage response (DDR) in a cyclophosphamide (CTX)-induced mouse model of premature ovarian failure (POF). METHODS: The POF model was established by injecting mice with CTX. The body, ovarian weights, the estrus cycle, and pathological changes of the ovaries were recorded. The serum levels of 17 ß-estradiol (E2) and follicle-stimulating hormone (FSH) were measured. The expression of Ki67, ß-galactosidase (ß-gal), p21, p53, γH2AX, and pATM in ovarian tissues was detected by immunohistochemistry. The expression of ß-gal, γH2AX, and pATM was analyzed by immunofluorescence staining of primary cultured granulosa cells (GCs). RESULTS: The body and ovarian weights decreased, the estrus cycles were erratic, and the FSH level increased, whereas the E2 level decreased in POF mice compared to controls. The pathological consequences of POF revealed an increase in atretic follicles, corpus luteum, and primordial follicles and a decrease in the number of primary, secondary, and tertiary follicles. Ki67 expression was reduced, ß-gal, p21, p53, γH2AX, and pATM expression were elevated in the ovaries of POF mice. The expression of ß-gal, γH2AX, and pATM increased in GCs with the concentration in a time-dependent manner. CONCLUSION: In total, CTX induced POF in mice, which was mediated by the DDR pathway of ATM-P53-P21.
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PURPOSE: To analyze the copy number variation (CNV) in the X-linked genes BCORL1, POF1B, and USP9X in idiopathic diminished ovarian reserve (DOR). METHODS: This case-control study included 47 women, 26 with DOR and 21 in the control group. Age, weight, height, BMI, and FSH level were evaluated, as well as antral follicle count (AFC), oocyte retrieval after controlled ovarian stimulation, and metaphase II (MII) oocytes. The CNVs of BCORL1, USP9X, and POF1B genes were measured by quantitative real time PCR (qPCR) using two reference genes, the HPRT1 (X-linked) and MFN2 (autosomal). Protein-protein interaction network and functional enrichment analysis were performed using the STRING database. RESULTS: The mean age was 36.52 ± 4.75 in DOR women and 35.38 ± 4.14 in control. Anthropometric measures did not differ between the DOR and control groups. DOR women presented higher FSH (p = 0.0025) and lower AFC (p < .0001), oocyte retrieval after COS (p = 0.0004), and MII oocytes (p < .0001) when compared to the control group. BCORL1 and POF1B did not differ in copy number between DOR and control. However, DOR women had more copies of USP9X than the control group (p = 0.028). CONCLUSION: The increase in the number of copies of the USP9X gene may lead to overexpression in idiopathic DOR and contribute to altered folliculogenesis and oocyte retrieval.
RESUMEN
Primary ovarian insufficiency (POI) is a common condition leading to the pathological decline of ovarian function in women of reproductive age, resulting in amenorrhea, hypogonadism, and infertility. Biochemical premature ovarian insufficiency (bPOI) is an intermediate stage in the pathogenesis of POI in which the fertility of patients has been reduced. Previous studies suggest that granulosa cells (GCs) play an essential role in the pathogenesis of POI, but their pathogenetic mechanisms remain unclear. To further explore the potential pathophysiological mechanisms of GCs in POI, we constructed a molecular long non-coding RNA (lncRNA)-microRNA (miRNA)-messenger RNA (mRNA) network using GC expression data collected from biochemical premature ovarian failure (bPOI) patients in the GEO database. We discovered that the GCs of bPOI patients had differential expression of 131 mRNAs, 191 lncRNAs, and 28 miRNAs. By systematic network analysis, we identified six key genes, including SRSF1, PDIA5, NEURL1B, UNK, CELF2, and CFL2, and five hub miRNAs, namely hsa-miR-27a-3p, hsa-miR-24-3p, hsa-miR-22-3p, hsa-miR-129-5p, and hsa-miR-17-5p, and the results suggest that the expression of these key genes may be regulated by two hub miRNAs, hsa-miR-27a-3p and hsa-miR-17-5p. Additionally, a POI model in vitro was created to confirm the expression of a few important genes. In this study, we discovered a unique lncRNA-miRNA-mRNA network based on the ceRNA mechanism in bPOI for the first time, and we screened important associated molecules, providing a partial theoretical foundation to better understand the pathogenesis of POI.
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MicroARNs , Insuficiencia Ovárica Primaria , ARN Largo no Codificante , Humanos , Femenino , ARN Largo no Codificante/genética , Insuficiencia Ovárica Primaria/genética , ARN Endógeno Competitivo , ARN Mensajero/genética , ARN Mensajero/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Células de la Granulosa/metabolismo , Redes Reguladoras de Genes/genética , Proteínas CELF/genética , Proteínas del Tejido Nervioso/genética , Factores de Empalme Serina-Arginina/genéticaRESUMEN
Small extracellular vesicles (sEVs) secreted by human umbilical cord have therapeutic effects on various degenerative diseases. However, the characteristics and potential functions of human umbilical cord mesenchymal stem cells (huMSCs)-derived sEVs, especially the role of premature ovarian failure (POF), are poorly understood. Here, we isolated and characterized huMSCs and their sEVs. huMSCs highly expressed CD73, CD90, and CD105. huMSC-sEVs showed typical exosomal features, highly expressing CD9, TSG101, and CD63. It was shown that huMSC-sEVs could be taken up by granulosa cells (GCs) and damaged ovarian tissue, which increased the levels of hormone secretion and reduced GCs apoptosis. We further confirmed that the levels of follicle-stimulating hormone in rat serum decreased dramatically, while the levels of estrogen (E2ï¼and anti-mullerian hormone (AMH) increased significantly with the treatment of huMSC-sEVs. Meanwhile, huMSC-sEVs treatment greatly reduced cell apoptosis and autophagy, while increased the phosphorylation levels of p-PI3K and p-Akt. Therefore, treatment with huMSC-sEVs significantly inhibited GCs apoptosis, improved ovarian morphology, promoted follicular development, inhibited follicular over-atresia, and improved ovarian reserve capacity in POF rats. Our study verified that activation of PI3K/Akt signaling pathway and regulation of cellular autophagy, thus reducing GCs death, are the mechanisms by which huMSC-sEVs restore ovarian tissue function.
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Apoptosis , Cisplatino , Vesículas Extracelulares , Células de la Granulosa , Células Madre Mesenquimatosas , Ovario , Insuficiencia Ovárica Primaria , Cordón Umbilical , Femenino , Células Madre Mesenquimatosas/efectos de los fármacos , Animales , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Humanos , Cordón Umbilical/citología , Insuficiencia Ovárica Primaria/inducido químicamente , Cisplatino/toxicidad , Apoptosis/efectos de los fármacos , Ratas , Ovario/efectos de los fármacos , Ovario/patología , Células de la Granulosa/efectos de los fármacos , Ratas Sprague-Dawley , Antineoplásicos/toxicidadRESUMEN
In this study, a mouse model of premature ovarian failure(POF) was constructed by injecting D-galactose(200 mg·kg~(-1)) into the back of the neck for 6 weeks. The mice were randomly divided into a normal group(group N), a model group(group M), and a Qiwei Guibao Granules group(group A, 12.87 g·kg~(-1)). Starting from the 11th day of modeling, group A was treated with Qiwei Guibao Granules by gavage for 32 days, while group M and group N were given equal volume of saline. Metabolomics analysis was used to explore the mechanism of action of Qiwei Guibao Granules in the treatment of POF. The results showed that compared with group N, the group M exhibited decreased wet weight of bilateral ovaries, increased levels of LH and FSH in serum, and significantly decreased levels of E_2 and PROG. After treatment with Qiwei Guibao Granules, compared with the group M, the group A showed a significant increase in the wet weight of bilateral ovaries, a significant decrease in the levels of FSH and LH in serum, and a significant increase in the level of E_2. Metabolomics analysis revealed 55 differential metabolites identified between group N and group M(14 upregulated and 41 downregulated compared with group N) and 82 differential metabolites identified between group M and group A(56 upregulated and 26 downregulated compared with group M), with 5 metabolites showing consistent changes between the group N vs group M. After excluding these 5 metabolites, 77 metabolites that changed after intervention with Qiwei Guibao Granules were focused on. These mainly involved histidine metabolism, glycine, serine, and threonine metabolism, and glycerophospholipid metabolism. Among them, carnosine, 1-methyl-L-histidine, imidazoleacetic acid, choline, L-threonine, beta-hydroxypyruvic acid, phosphatidylcholine, and glycerol-3-phosphate were the major differential metabolites in these three metabolic pathways. Therefore, Qiwei Guibao Granules may exert therapeutic effects on POF mice by regulating amino acid metabolism and lipid metabolism in the mouse body.