RESUMEN
Molecular mechanisms of ovarian aging and female age-related fertility decline remain unclear. We surveyed the single-cell transcriptomic landscape of ovaries from young and aged non-human primates (NHPs) and identified seven ovarian cell types with distinct gene-expression signatures, including oocyte and six types of ovarian somatic cells. In-depth dissection of gene-expression dynamics of oocytes revealed four subtypes at sequential and stepwise developmental stages. Further analysis of cell-type-specific aging-associated transcriptional changes uncovered the disturbance of antioxidant signaling specific to early-stage oocytes and granulosa cells, indicative of oxidative damage as a crucial factor in ovarian functional decline with age. Additionally, inactivated antioxidative pathways, increased reactive oxygen species, and apoptosis were observed in granulosa cells from aged women. This study provides a comprehensive understanding of the cell-type-specific mechanisms underlying primate ovarian aging at single-cell resolution, revealing new diagnostic biomarkers and potential therapeutic targets for age-related human ovarian disorders.
Asunto(s)
Envejecimiento/genética , Ovario/fisiología , Análisis de la Célula Individual/métodos , Transcriptoma , Anciano , Animales , Antioxidantes/metabolismo , Apoptosis/fisiología , Atlas como Asunto , Biomarcadores , Línea Celular Tumoral , Femenino , Células de la Granulosa/metabolismo , Humanos , Macaca fascicularis , Oocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Cell fate specification by lateral inhibition typically involves contact signaling through the Delta-Notch signaling pathway. However, whether this is the only signaling mode mediating lateral inhibition remains unclear. Here we show that in zebrafish oogenesis, a group of cells within the granulosa cell layer at the oocyte animal pole acquire elevated levels of the transcriptional coactivator TAZ in their nuclei. One of these cells, the future micropyle precursor cell (MPC), accumulates increasingly high levels of nuclear TAZ and grows faster than its surrounding cells, mechanically compressing those cells, which ultimately lose TAZ from their nuclei. Strikingly, relieving neighbor-cell compression by MPC ablation or aspiration restores nuclear TAZ accumulation in neighboring cells, eventually leading to MPC re-specification from these cells. Conversely, MPC specification is defective in taz-/- follicles. These findings uncover a novel mode of lateral inhibition in cell fate specification based on mechanical signals controlling TAZ activity.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Oogénesis/fisiología , Proteínas de Pez Cebra/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Linaje de la Célula , Núcleo Celular/metabolismo , Femenino , Células de la Granulosa/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Oocitos/metabolismo , Oocitos/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiología , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Pez Cebra/metabolismo , Proteínas de Pez Cebra/antagonistas & inhibidoresRESUMEN
Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.
Asunto(s)
Linaje de la Célula , Células Germinativas , Ovario , Diferenciación Sexual , Análisis de la Célula Individual , Testículo , Animales , Cromatina/genética , Cromatina/metabolismo , Femenino , Células Germinativas/citología , Células Germinativas/metabolismo , Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Humanos , Inmunoglobulinas , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana , Proteínas de la Membrana , Ratones , Microscopía Fluorescente , Ovario/citología , Ovario/embriología , Factor de Transcripción PAX8 , Embarazo , Primer Trimestre del Embarazo , Segundo Trimestre del Embarazo , Receptores Inmunológicos , Diferenciación Sexual/genética , Testículo/citología , Testículo/embriología , TranscriptomaRESUMEN
Ovulation is essential for reproductive success, yet the underlying cellular and molecular mechanisms are far from clear. Here, we applied high-resolution spatiotemporal transcriptomics to map out cell type- and ovulation stage-specific molecular programs as function of time during follicle maturation and ovulation in mice. Our analysis revealed dynamic molecular transitions within granulosa cell types that occur in tight coordination with mesenchymal cell proliferation. We identified molecular markers for the emerging cumulus cell fate during the preantral-to-antral transition. We describe transcriptional programs that respond rapidly to ovulation stimulation and those associated with follicle rupture, highlighting the prominent roles of apoptotic and metabolic pathways during the final stages of follicle maturation. We further report stage-specific oocyte-cumulus cell interactions and diverging molecular differentiation in follicles approaching ovulation. Collectively, this study provides insights into the cellular and molecular processes that regulate mouse ovarian follicle maturation and ovulation with important implications for advancing therapeutic strategies in reproductive medicine.
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Ascomicetos , Ovario , Femenino , Animales , Ratones , Ovulación , Folículo Ovárico , Reproducción , Células de la GranulosaRESUMEN
The dynamic transcriptional regulation and interactions of human germlines and surrounding somatic cells during folliculogenesis remain unknown. Using RNA sequencing (RNA-seq) analysis of human oocytes and corresponding granulosa cells (GCs) spanning five follicular stages, we revealed unique features in transcriptional machinery, transcription factor networks, and reciprocal interactions in human oocytes and GCs that displayed developmental-stage-specific expression patterns. Notably, we identified specific gene signatures of two cell types in particular developmental stage that may reflect developmental competency and ovarian reserve. Additionally, we uncovered key pathways that may concert germline-somatic interactions and drive the transition of primordial-to-primary follicle, which represents follicle activation. Thus, our work provides key insights into the crucial features of the transcriptional regulation in the stepwise folliculogenesis and offers important clues for improving follicle recruitment in vivo and restoring fully competent oocytes in vitro.
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Comunicación Celular/genética , Células de la Granulosa/fisiología , Oocitos/fisiología , Folículo Ovárico/fisiología , Reserva Ovárica/genética , Transcriptoma , Adulto , Animales , Biología Computacional , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Ratones , Folículo Ovárico/citología , Transducción de Señal/genética , Análisis de la Célula Individual , Especificidad de la Especie , Transcripción Genética , Adulto JovenRESUMEN
The ovarian follicle reserve, formed pre- or perinatally, comprises all oocytes for lifetime reproduction. Depletion of this reserve results in infertility. Steroidogenic factor 1 (SF-1; Nr5a1) and liver receptor homolog 1 (LRH-1; Nr5a2) are two orphan nuclear receptors that regulate adult endocrine function, but their role in follicle formation is unknown. We developed models of conditional depletion of SF-1 or LRH-1 from prenatal ovaries. Depletion of SF-1, but not LRH-1, resulted in dramatically smaller ovaries and fewer primordial follicles. This was mediated by increased oocyte death, resulting from increased ovarian inflammation and increased Notch signaling. Major dysregulated genes were Iroquois homeobox 3 and 5 and their downstream targets involved in the establishment of the ovarian laminin matrix and oocyte-granulosa cell gap junctions. Disruptions of these pathways resulted in follicles with impaired basement membrane formation and compromised oocyte-granulosa communication networks, believed to render them more prone to atresia. This study identifies SF-1 as a key regulator of the formation of the ovarian reserve.
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Reserva Ovárica , Embarazo , Femenino , Humanos , Factor Esteroidogénico 1/genética , Factor Esteroidogénico 1/metabolismo , Reserva Ovárica/genética , Folículo Ovárico/metabolismo , Ovario/metabolismo , Células de la Granulosa/metabolismoRESUMEN
Severe hypoxia induced by vascular compromise (ovarian torsion, surgery), obliteration of vessels (aging, chemotherapy, particularly platinum drugs) can cause massive follicle atresia. On the other hand, hypoxia increases the occurrence of DNA double-strand breaks (DSBs) and triggers cellular damage repair mechanisms; however, if the damage is not promptly repaired, it can also induce the apoptosis program. Insulin-like growth factor-I (IGF-I) is a polypeptide hormone that plays essential roles in stimulating mammalian follicular development. Here, we report a novel role for IGF-I in protecting hypoxic GCs from apoptosis by promoting DNA repair through the homologous recombination (HR) process. Indeed, the hypoxic environment within follicles significantly inhibited the efficiency of HR-directed DNA repair. The presence of IGF-I-induced HR pathway to alleviate hypoxia-induced DNA damage and apoptosis primarily through upregulating the expression of the RAD51 recombinase. Importantly, we identified a new transcriptional regulator of RAD51, namely E2F8, which mediates the protective effects of IGF-I on hypoxic GCs by facilitating the transcriptional activation of RAD51. Furthermore, we demonstrated that the PI3K/AKT pathway is crucial for IGF-I-induced E2F8 expression, resulting in increased RAD51 expression and enhanced HR activity, which mitigates hypoxia-induced DNA damage and thereby protects against GCs apoptosis. Together, these findings define a novel mechanism of IGF-I-mediated GCs protection by activating the HR repair through the PI3K/AKT/E2F8/RAD51 pathway under hypoxia.
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Proteínas Proto-Oncogénicas c-akt , Reparación del ADN por Recombinación , Femenino , Animales , Porcinos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Reparación del ADN , Recombinación Homóloga , Recombinasa Rad51/genética , Hipoxia , Células de la Granulosa/metabolismo , Apoptosis , Mamíferos/metabolismoRESUMEN
Mammalian oocytes go through a long and complex developmental process while acquiring the competencies that are required for fertilization and embryogenesis. Recent advances in molecular genetics and quantitative live imaging reveal new insights into the molecular basis of the communication between the oocyte and ovarian somatic cells as well as the dynamic cytoskeleton-based events that drive each step along the pathway to maturity. Whereas self-organization of microtubules and motor proteins direct meiotic spindle assembly for achieving genome reduction, actin filaments are instrumental for spindle positioning and the establishment of oocyte polarity needed for extrusion of polar bodies. Meiotic chromatin provides key instructive signals while being 'chauffeured' by both cytoskeletal systems.
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Citoesqueleto de Actina/fisiología , Células de la Granulosa/fisiología , Meiosis , Oocitos/citología , Oocitos/fisiología , Oogénesis , Folículo Ovárico/citología , Citoesqueleto de Actina/ultraestructura , Animales , Comunicación Celular , Cromatina/fisiología , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/fisiología , Desarrollo Embrionario , Femenino , Humanos , Meiosis/genética , Ratones , Microtúbulos/metabolismo , Oocitos/crecimiento & desarrollo , Folículo Ovárico/fisiologíaRESUMEN
In females, the pathophysiological mechanism of poor ovarian response (POR) is not fully understood. Considering the expression level of p62 was significantly reduced in the granulosa cells (GCs) of POR patients, this study focused on identifying the role of the selective autophagy receptor p62 in conducting the effect of follicle-stimulating hormone (FSH) on antral follicles (AFs) formation in female mice. The results showed that p62 in GCs was FSH responsive and that its level increased to a peak and then decreased time-dependently either in ovaries or in GCs after gonadotropin induction in vivo. GC-specific deletion of p62 resulted in subfertility, a significantly reduced number of AFs and irregular estrous cycles, which were same as pathophysiological symptom of POR. By conducting mass spectrum analysis, we found the ubiquitination of proteins was decreased, and autophagic flux was blocked in GCs. Specifically, the level of nonubiquitinated Wilms tumor 1 homolog (WT1), a transcription factor and negative controller of GC differentiation, increased steadily. Co-IP results showed that p62 deletion increased the level of ubiquitin-specific peptidase 5 (USP5), which blocked the ubiquitination of WT1. Furthermore, a joint analysis of RNA-seq and the spatial transcriptome sequencing data showed the expression of steroid metabolic genes and FSH receptors pivotal for GCs differentiation decreased unanimously. Accordingly, the accumulation of WT1 in GCs deficient of p62 decreased steroid hormone levels and reduced FSH responsiveness, while the availability of p62 in GCs simultaneously ensured the degradation of WT1 through the ubiquitinâproteasome system and autophagolysosomal system. Therefore, p62 in GCs participates in GC differentiation and AF formation in FSH induction by dynamically controlling the degradation of WT1. The findings of the study contributes to further study the pathology of POR.
Asunto(s)
Hormona Folículo Estimulante , Células de la Granulosa , Folículo Ovárico , Proteína Sequestosoma-1 , Ubiquitinación , Proteínas WT1 , Animales , Hormona Folículo Estimulante/metabolismo , Hormona Folículo Estimulante/farmacología , Femenino , Proteínas WT1/metabolismo , Proteínas WT1/genética , Ratones , Folículo Ovárico/metabolismo , Folículo Ovárico/efectos de los fármacos , Células de la Granulosa/metabolismo , Células de la Granulosa/efectos de los fármacos , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Ratones Endogámicos C57BL , Autofagia/efectos de los fármacos , Proteolisis/efectos de los fármacos , Humanos , Ratones NoqueadosRESUMEN
Abnormal autophagy is one of the vital features in polycystic ovary syndrome (PCOS). However, the underlying molecular mechanisms remain unelucidated. In this study, we aimed to investigate whether Block of Proliferation 1 (BOP1) is involved in the onset of autophagy activation of granulosa cells in PCOS. Firstly, we found that BOP1 expression was significantly down-regulated in the ovaries of PCOS mice, which was associated with the development of PCOS. Next, local injection of lentiviral vectors in the ovary for the overexpression of BOP1 significantly alleviated the phenotypes of elevated androgens, disturbed estrous cycle, and abnormal follicular development in PCOS mice. Subsequently, we found that knockdown of BOP1 activated autophagy of granulosa cells in the in vitro experiments, whereas overexpression of BOP1 inhibited autophagy in both in vivo and in vitro models. Mechanistically, BOP1 knockdown triggered the nucleolus stress response, which caused RPL11 to be released from the nucleolus into the nucleoplasm and inhibited the E3 ubiquitination ligase of MDM2, thereby enhancing the stability of p53. Subsequently, P53 inhibited mTOR, thereby activating autophagy in granulosa cells. In addition, the mRNA level of BOP1 was negatively correlated with antral follicle count (AFC), body-mass index (BMI), serum androgen levels, and anti-Mullerian hormone (AMH) in patients with PCOS. In summary, our study demonstrates that BOP1 downregulation inhibits mTOR phosphorylation through activation of the p53-dependent nucleolus stress response, which subsequently contributes to aberrant autophagy in granulosa cells, revealing that BOP1 may be a key target for probing the mechanisms of PCOS.
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Síndrome del Ovario Poliquístico , Animales , Femenino , Humanos , Ratones , Células de la Granulosa/metabolismo , Folículo Ovárico/metabolismo , Síndrome del Ovario Poliquístico/genética , Síndrome del Ovario Poliquístico/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Supporting cells of the ovary, termed granulosa cells, are essential for ovarian differentiation and oogenesis by providing a nurturing environment for oocyte maintenance and maturation. Granulosa cells are specified in the fetal and perinatal ovary, and sufficient numbers of granulosa cells are critical for the establishment of follicles and the oocyte reserve. Identifying the cellular source from which granulosa cells and their progenitors are derived is an integral part of efforts to understand basic ovarian biology and the etiology of female infertility. In particular, the contribution of mesenchymal cells, especially perivascular cells, to ovarian development is poorly understood but is likely to be a source of new information regarding ovarian function. Here we have identified a cell population in the fetal ovary, which is a Nestin-expressing perivascular cell type. Using lineage tracing and ex vivo organ culture methods, we determined that perivascular cells are multipotent progenitors that contribute to granulosa, thecal, and pericyte cell lineages in the ovary. Maintenance of these progenitors is dependent on ovarian vasculature, likely reliant on endothelial-mesenchymal Notch signaling interactions. Depletion of Nestin+ progenitors resulted in a disruption of granulosa cell specification and in an increased number of germ cell cysts that fail to break down, leading to polyovular ovarian follicles. These findings highlight a cell population in the ovary and uncover a key role for vasculature in ovarian differentiation, which may lead to insights into the origins of female gonad dysgenesis and infertility.
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Ovario , Pericitos , Animales , Femenino , Células de la Granulosa/metabolismo , Nestina/genética , Nestina/metabolismo , Oogénesis/fisiología , Folículo Ovárico , Ovario/metabolismoRESUMEN
The follicle is the basic structural and functional unit of the ovary in female mammals. The excessive depletion of follicles will lead to diminished ovarian reserve or even premature ovarian failure, resulting in diminished ovarian oogenesis and endocrine function. Excessive follicular depletion is mainly due to loss of primordial follicles. Our analysis of published human ovarian single-cell sequencing results by others revealed a significant increase in rho-associated protein kinase 1 (ROCK1) expression during primordial follicle development. However, the role of ROCK1 in primordial follicle development and maintenance is not clear. This study revealed a gradual increase in ROCK1 expression during primordial follicle activation. Inhibition of ROCK1 resulted in reduced primordial follicle activation, decreased follicular reserve, and delayed development of growing follicles. This effect may be achieved through the HIPPO pathway. The present study indicates that ROCK1 is a key molecule for primordial follicular reserve and follicular development.NEW & NOTEWORTHY ROCK1, one of the Rho GTPases, plays an important role in primordial follicle reserve and follicular development. ROCK1 was primarily expressed in the cytoplasm of oocytes and granulosa cell in mice. Inhibition of ROCK1 significantly reduced the primordial follicle reserve and delayed growing follicle development. ROCK1 regulates primordial follicular reserve and follicle development through the HIPPO signaling pathway. These findings shed new lights on the physiology of sustaining female reproduction.
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Oocitos , Folículo Ovárico , Animales , Femenino , Humanos , Ratones , Células de la Granulosa/metabolismo , Mamíferos , Oogénesis , Folículo Ovárico/metabolismo , Ovario/metabolismo , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
Oxidative stress triggered by aging, radiation, or inflammation impairs ovarian function by inducing granulosa cell (GC) apoptosis. However, the mechanism inducing GC apoptosis has not been characterized. Here, we found that ovarian GCs from aging patients showed increased oxidative stress, enhanced reactive oxygen species activity, and significantly decreased expression of the known antiapoptotic factor sphingosine-1-phosphate/sphingosine kinase 1 (SPHK1) in GCs. Interestingly, the expression of Krüppel-like factor 12 (KLF12) was significantly increased in the ovarian GCs of aging patients. Furthermore, we determined that KLF12 was significantly upregulated in hydrogen peroxide-treated GCs and a 3-nitropropionic acid-induced in vivo model of ovarian oxidative stress. This phenotype was further confirmed to result from inhibition of SPHK1 by KLF12. Interestingly, when endogenous KLF12 was knocked down, it rescued oxidative stress-induced apoptosis. Meanwhile, supplementation with SPHK1 partially reversed oxidative stress-induced apoptosis. However, this function was lost in SPHK1 with deletion of the binding region to the KLF12 promoter. SPHK1 reversed apoptosis caused by hydrogen peroxide-KLF12 overexpression, a result further confirmed in an in vitro ovarian culture model and an in vivo 3-nitropropionic acid-induced ovarian oxidative stress model. Overall, our study reveals that KLF12 is involved in regulating apoptosis induced by oxidative stress in aging ovarian GCs and that sphingosine-1-phosphate/SPHK1 can rescue GC apoptosis by interacting with KLF12 in negative feedback.
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Envejecimiento , Apoptosis , Células de la Granulosa , Peróxido de Hidrógeno , Factores de Transcripción de Tipo Kruppel , Lisofosfolípidos , Fosfotransferasas (Aceptor de Grupo Alcohol) , Esfingosina , Femenino , Humanos , Envejecimiento/metabolismo , Retroalimentación Fisiológica , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Peróxido de Hidrógeno/farmacología , Técnicas In Vitro , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/biosíntesis , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Lisofosfolípidos/biosíntesis , Lisofosfolípidos/metabolismo , Técnicas de Cultivo de Órganos , Estrés Oxidativo/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Regiones Promotoras Genéticas , Esfingosina/biosíntesis , Esfingosina/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Lack of estradiol production by granulosa cells blocks follicle development, causes failure of estrous initiation, and results in an inability to ovulate. The ubiquitin-proteasome system plays a critical role in maintaining protein homeostasis and stability of the estrous cycle, but knowledge of deubiquitination enzyme function in estradiol synthesis is limited. Here, we observe that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is more significant in estrous sows and high litter-size sows than in nonestrous sows and low-yielding sows. Overexpression of UCHL1 promotes estradiol synthesis in granulosa cells, and interference with UCHL1 has the opposite effect. UCHL1 binds, deubiquitinates, and stabilizes voltage-dependent anion channel 2 (VDAC2), promoting the synthesis of the estradiol precursor pregnenolone. Cysteine 90 (C90) of UCHL1 is necessary for its deubiquitination activity, and Lys45 and Lys64 in VDAC2 are essential for its ubiquitination and degradation. In vivo, compared with WT and sh-NC-AAV groups, the estrus cycle of female mice is disturbed, estradiol level is decreased, and the number of antral follicles is decreased after the injection of sh-UCHL1-AAV into ovarian tissue. These findings suggest that UCHL1 promotes estradiol synthesis by stabilizing VDAC2 and identify UCHL1 as a candidate gene affecting reproductive performance.
Asunto(s)
Estradiol , Ubiquitina Tiolesterasa , Canal Aniónico 2 Dependiente del Voltaje , Animales , Femenino , Ratones , Células de la Granulosa/metabolismo , Folículo Ovárico/metabolismo , Porcinos , Ubiquitina Tiolesterasa/metabolismo , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Sus scrofaRESUMEN
To explore whether granulosa cell (GC)-derived exosomes (GC-Exos) and follicular fluid-derived exosomes (FF-Exos) have functional similarities in follicle development and to establish relevant experiments to validate whether GC-Exos could serve as a potential substitute for follicular fluid-derived exosomes to improve folliculogenesis. GC-Exos were characterized. MicroRNA (miRNA) profiles of exosomes from human GCs and follicular fluid were analyzed in depth. The signature was associated with folliculogenesis, such as phosphatidylinositol 3 kinases-protein kinase B signal pathway, mammalian target of rapamycin signal pathway, mitogen-activated protein kinase signal pathway, Wnt signal pathway, and cyclic adenosine monophosphate signal pathway. A total of five prominent miRNAs were found to regulate the above five signaling pathways. These miRNAs include miRNA-486-5p, miRNA-10b-5p, miRNA-100-5p, miRNA-99a-5p, and miRNA-21-5p. The exosomes from GCs and follicular fluid were investigated to explore the effect on folliculogenesis by injecting exosomes into older mice. The proportion of follicles at each stage is counted to help us understand folliculogenesis. Exosomes derived from GCs were isolated successfully. miRNA profiles demonstrated a remarkable overlap between the miRNA profiles of FF-Exos and GC-Exos. The shared miRNA signature exhibited a positive influence on follicle development and activation. Furthermore, exosomes derived from GCs and follicular fluid promoted folliculogenesis in older female mice. Exosomes derived from GCs had similar miRNA profiles and follicle-promoting functions as follicular fluid exosomes. Consequently, GC-Exos are promising for replacing FF-Exos and developing new commercial reagents to improve female fertility.
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Exosomas , Células de la Granulosa , MicroARNs , Folículo Ovárico , Animales , Femenino , Humanos , Ratones , Exosomas/genética , Exosomas/metabolismo , Líquido Folicular/metabolismo , Células de la Granulosa/metabolismo , MicroARNs/genética , Folículo Ovárico/metabolismo , Transducción de SeñalRESUMEN
Follicle selection in hens refers to a biological process that only one small yellow follicle (SYF) is selected daily or near-daily for following hierarchical development (from F5/F6 to F1) until ovulation. MFN2 is a kind of GTPases located on the mitochondrial outer membrane, which plays a crucial role in mitochondrial fusion. This study aimed to elucidate the role of MFN2 in proliferation and progesterone biosynthesis of granulosa cells (GCs) during follicle selection in hens. The results showed that GCs began to produce progesterone (P4) after follicle selection, accompanied with changes from multi-layer with flat cells to single layer with cubic cells. MFN2 was detected in GCs of follicles from SYF to F1. After follicle selection, the expression level of MFN2 in GCs upregulated significantly, accompanied with increases in P4 biosynthesis, ATP production, mitochondrial DNA (mtDNA) copy numbers of granulosa cells. FSH (80 ng/mL) facilitated the effects of P4 biosynthesis and secretion, ATP production, mtDNA copy numbers, cell proliferation and the MFN2 transcription of granulosa cells from F5 (F5G) in vitro. However, FSH treatment did not promote P4 secretion in granulosa cells from SYF (SYFG) in vitro. Meanwhile, we observed that change fold of MFN2 transcription, ATP production, mtDNA copy numbers and cell proliferation rate in F5G after treatment with FSH were greater than those in SYFG. Furthermore, expression levels of MFN2 protein and messenger RNA in F5G were significantly higher than those in SYFG after treatment with FSH. P4 biosynthesis, ATP production, mtDNA copy numbers as well as cell proliferation reduced significantly in F5G with MFN2 knockdown. Oppositely, P4 biosynthesis, ATP production, mtDNA copy numbers and cell proliferation increased significantly in SYFG after the overexpression of MFN2. Our results suggest that the upregulation of MFN2 may be involved in the initiation of P4 biosynthesis, and promotion of GCs proliferation during follicle selection.
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Hormona Luteinizante , Progesterona , Femenino , Animales , Progesterona/metabolismo , Pollos/genética , Células de la Granulosa/metabolismo , Hormona Folículo Estimulante/farmacología , ADN Mitocondrial/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
Follicular cysts are a common reproductive disorder in domestic animals that cause considerable economic losses to the farming industry. Effective prevention and treatment methods are lacking because neither the pathogenesis nor formation mechanisms of follicular cysts are well-understood. In this study, we first investigated the granulosa cells (GCs) of cystic follicles isolated from pigs. We observed a significant reduction in the expression of methyltransferase-like 3 (METTL3). Subsequent experiments revealed that METTL3 downregulation in GCs caused a decrease in m6A modification of pri-miR-21. This reduction further inhibited DGCR8 recognition and binding to pri-miR-21, dampening the synthesis of mature miR-21-5p. Additionally, the decrease in miR-21-5p promotes IL-1ß expression in GCs. Elevated IL-1ß activates the NFκB pathway, in turn upregulating apoptotic genes TNFa and BAX/BCL2. The subsequent apoptosis of GCs and inhibition of autophagy causes downregulation of CYP19A1 expression. These processes lower oestrogen secretion and contribute to follicular cyst formation. In conclusion, our findings provide a foundation for understanding and further exploring the mechanisms of follicular-cyst development in farm animals. This work has important implications for treating ovarian disorders in livestock and could potentially be extended to humans.
Asunto(s)
Apoptosis , Células de la Granulosa , Metiltransferasas , MicroARNs , Animales , Femenino , Apoptosis/genética , Células Cultivadas , Regulación hacia Abajo , Quiste Folicular/genética , Quiste Folicular/patología , Quiste Folicular/metabolismo , Células de la Granulosa/metabolismo , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , MicroARNs/genética , MicroARNs/metabolismo , FN-kappa B/metabolismo , FN-kappa B/genética , Transducción de Señal , Porcinos , Proteínas de Unión al ARN/metabolismoRESUMEN
The developmental fate of ovarian follicles is primarily determined by the survival status (proliferation or apoptosis) of granulosa cells (GCs). Owing to the avascular environment within follicles, GCs are believed to live in a hypoxic niche. Follicle-stimulating hormone (FSH) has been reported to improve GCs survival by governing hypoxia-inducible factor-1α (HIF-1α)-dependent hypoxia response, but the underlying mechanisms remain poorly understood. Growth arrest-specific gene 6 (GAS6) is a secreted ligand of tyrosine kinase receptors, and has been documented to facilitate tumor growth. Here, we showed that the level of GAS6 was markedly increased in mouse ovarian GCs after the injection of FSH. Specifically, FSH-induced GAS6 expression was accompanied by HIF-1α accumulation under conditions of hypoxia both in vivo and in vitro, whereas inhibition of HIF-1α with small interfering RNAs/antagonist repressed both expression and secretion of GAS6. As such, Luciferase reporter assay and chromatin immunoprecipitation assay showed that HIF-1α directly bound to a hypoxia response element site within the Gas6 promoter and contributed to the regulation of GAS6 expression in response to FSH. Notably, blockage of GAS6 and/or its receptor Axl abrogated the pro-survival effects of FSH under hypoxia. Moreover, phosphorylation of Axl by GAS6 is required for FSH-mediated Akt activation and the resultant pro-survival phenotypes. Finally, the in vitro findings were verified in vivo, which showed that FSH-induced proliferative and antiapoptotic effects in ovarian GCs were diminished after blocking GAS6/Axl using HIF-1α antagonist. These findings highlight a novel function of FSH in preserving GCs viability against hypoxic stress by activating the HIF-1a-GAS6-Axl-Akt pathway.
Asunto(s)
Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Animales , Femenino , Ratones , Hormona Folículo Estimulante/farmacología , Células de la Granulosa/metabolismo , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratones Endogámicos ICRRESUMEN
BACKGROUND: Neddylation, an important post-translational modification (PTM) of proteins, plays a crucial role in follicular development. MLN4924 is a small-molecule inhibitor of the neddylation-activating enzyme (NAE) that regulates various biological processes. However, the regulatory mechanisms of neddylation in rabbit ovarian cells have not been emphasized. Here, the transcriptome and metabolome profiles in granulosa cells (GCs) treated with MLN4924 were utilized to identify differentially expressed genes, followed by pathway analysis to precisely define the altered metabolisms. RESULTS: The results showed that 563 upregulated and 910 downregulated differentially expressed genes (DEGs) were mainly enriched in pathways related to cancer, cell cycle, PI3K-AKT, progesterone-mediated oocyte maturation, and PPAR signaling pathway. Furthermore, we characterized that MLN4924 inhibits PPAR-mediated lipid metabolism, and disrupts the cell cycle by promoting the apoptosis and proliferation of GCs. Importantly, we found the reduction of several metabolites in the MLN4924 treated GCs, including glycerophosphocholine, arachidic acid, and palmitic acid, which was consistent with the deregulation of PPAR signaling pathways. Furthermore, the increased metabolites included 6-Deoxy-6-sulfo-D-glucono-1,5-lactone and N-Acetyl-D-glucosaminyldiphosphodolichol. Combined with transcriptome data analyses, we identified genes that strongly correlate with metabolic dysregulation, particularly those related to glucose and lipid metabolism. Therefore, neddylation inhibition may disrupt the energy metabolism of GCs. CONCLUSIONS: These results provide a foundation for in-depth research into the role and molecular mechanism of neddylation in ovary development.
Asunto(s)
Ciclopentanos , Receptores Activados del Proliferador del Peroxisoma , Fosfatidilinositol 3-Quinasas , Pirimidinas , Femenino , Animales , Conejos , Células de la Granulosa , Metabolismo de los LípidosRESUMEN
Cumulus granulosa cells (CGCs) play a crucial role in follicular development, but so far, no research has explored the impact of SARS-CoV-2 infection on ovarian function from the perspective of CGCs. In the present study, we compared the cycle outcomes between infected and uninfected female patients undergoing controlled ovarian stimulation, performed bulk RNA-sequencing of collected CGCs, and used bioinformatic methods to explore transcriptomic changes. The results showed that women with SARS-CoV-2 infection during stimulation had significantly lower number of oocytes retrieved and follicle-oocyte index, while subsequent fertilization and embryo development were similar. CGCs were not directly infected by SARS-CoV-2, but exhibited dramatic differences in gene expression (156 up-regulated and 65 down-regulated). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses demonstrated a high enrichment in antiviral, immune and inflammatory responses with necroptosis. In addition, the pathways related to telomere organization and double strand break repair were significantly affected by infection in gene set enrichment analysis. Further weighted gene co-expression network analysis identified a key module associated with ovarian response traits, which was mainly enriched as a decrease of leukocyte chemotaxis and migration in CGCs. For the first time, our study describes how SARS-CoV-2 infection indirectly affects CGCs at the transcriptional level, which may impair oocyte-CGC crosstalk and consequently lead to poor ovarian response during fertility treatment.