RESUMO
In mammalian females, the transition from dormancy in primordial follicles to follicular development is critical for maintaining ovarian function and reproductive longevity. In mice, the quiescent primary oocyte of the primordial follicle contains a Balbiani body (B-body), an organelle aggregate comprised of a spherical structure of Golgi complexes. Here we show that the structure of the B-body is maintained by microtubules and actin. The B-body stores mRNA-capping enzyme and 597 mRNAs associated with mRNA-decapping enzyme 1 A (DCP1A). Gene ontology analysis results indicate that proteins encoded by these mRNAs function in enzyme binding, cellular component organization and packing of telomere ends. Pharmacological depolymerization of microtubules or actin led to B-body disassociation and nascent protein synthesis around the dissociated B-bodies within three hours. An increased number of activated developing follicles were observed in ovaries with prolonged culture and the in vivo mouse model. Our results indicate that the mouse B-body is involved in the activation of dormant primordial follicles likely via translation of the B-body-associated RNAs in primary oocytes.
Assuntos
Oócitos , Folículo Ovariano , Animais , Oócitos/metabolismo , Camundongos , Feminino , Folículo Ovariano/metabolismo , Folículo Ovariano/citologia , RNA/metabolismo , RNA/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Microtúbulos/metabolismo , Actinas/metabolismo , Actinas/genética , Complexo de Golgi/metabolismoRESUMO
Oocytes play a crucial role in transmitting maternal mitochondrial DNA (mtDNA), essential for the continuation of species. However, the effects of mitochondrial reactive oxygen species (ROS) on mammalian oocyte maturation and mtDNA maintenance remain unclear. We investigated this by conditionally knocking out the Sod2 gene in primordial follicles, elevating mitochondrial matrix ROS levels from early oocyte stages. Our data indicates that reproductive aging in Sod2 conditional knockout females begins at 6 months, with oxidative stress impairing oocyte quality, particularly affecting OXPHOS complex II and mtDNA-encoded mRNA levels. Despite unchanged mtDNA mutation load, mtDNA copy numbers exhibited significant variations. Strikingly, reducing mtDNA copy numbers by reducing mtSSB protein, crucial for mtDNA replication, accelerated reproductive aging onset to three months, underscoring the critical role of mtDNA copy number maintenance under oxidative stress conditions. This research provides new insights into the relationship among mitochondrial ROS, mtDNA, and reproductive aging, offering potential strategies for delaying aging-related fertility decline.
Assuntos
Envelhecimento , DNA Mitocondrial , Oócitos , Espécies Reativas de Oxigênio , Animais , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Oócitos/metabolismo , Feminino , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento/genética , Camundongos , Reprodução/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Estresse Oxidativo , Camundongos Knockout , Variações do Número de Cópias de DNA , Mitocôndrias/metabolismo , Mitocôndrias/genéticaRESUMO
Context In vitro embryo production in pigs is an important tool for advancing biomedical research. Intracytoplasmic sperm injection (ICSI) circumvents the polyspermy problems associated with conventional IVF in porcine. However, the suboptimal efficiency for ICSI in pigs requires new strategies to increase blastocyst formation rates. Aim To investigate novel methods for assisted activation using the zinc chelator 1,10-phenanthroline (PHEN), and to improve embryo developmental competence and quality of ICSI porcine blastocyst. Methods ICSI embryos were treated with PHEN after or before sperm injection, recording pronuclear formation, blastocyst rate and the expression of SMARCA4, OCT4, SOX2 and CDX2. Key results Neither electrical nor PHEN significantly improves pronuclear formation rates before or after ICSI. Following in vitro culture to the blastocyst stage, no significant differences were observed in developmental rates among the groups. Moreover, the use of PHEN did not alter the total cell number or the expression of OCT4, SOX2 and CDX2 in pig ICSI blastocysts. Conclusions Assisted oocyte activation with PHEN does not affect the preimplantation development of ICSI-derived pig embryos. Implications These results hold significance in refining and advancing the application of assisted oocyte activation techniques. They offer insights into addressing fertility issues and propelling advancements in human and animal reproductive medicine.
Assuntos
Quelantes , Desenvolvimento Embrionário , Oócitos , Fenantrolinas , Injeções de Esperma Intracitoplásmicas , Animais , Injeções de Esperma Intracitoplásmicas/veterinária , Injeções de Esperma Intracitoplásmicas/métodos , Suínos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Fenantrolinas/farmacologia , Feminino , Quelantes/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Zinco/farmacologia , Técnicas de Cultura Embrionária/veterinária , MasculinoRESUMO
Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes. The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system, where most males initially develop juvenile ovaries before committing to male fate. In species with chromosomal sex determination, gonadal somatic cells are recognized as key determinants of germ cell fate. Notably, the loss of germ cells in zebrafish leads to masculinization, implying that germ cells harbor an intrinsic feminization signal. However, the specific signal triggering oogenesis in zebrafish remains unclear. In the present study, we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells. Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males. Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis, leading to premature spermatogenesis during early gonadal development. Furthermore, while mutation of the pro-male gene dmrt1 led to fertile female differentiation, simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis, with a large proportion of germ cells arrested as germline stem cells, highlighting the crucial role of foxl2l in oogenesis. Overall, this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.
Assuntos
Oogênese , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Oogênese/fisiologia , Oogênese/genética , Feminino , Masculino , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Células Germinativas/fisiologia , Proteína Forkhead Box L2/genética , Proteína Forkhead Box L2/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Espermatogênese/fisiologia , Espermatogênese/genética , Oócitos/fisiologiaRESUMO
G-protein-coupled receptor kinase 2 (GRK2) interacts with Gßγ and Gαq, subunits of G-protein alpha, to regulate cell signalling. The second messenger inositol trisphosphate, produced by activated Gαq, promotes calcium release from the endoplasmic reticulum (ER) and regulates maturation-promoting factor (MPF) activity. This study aimed to investigate the role of GRK2 in MPF activity during the meiotic maturation of porcine oocytes. A specific inhibitor of GRK2 (ßi) was used in this study. The present study showed that GRK2 inhibition increased the percentage of oocyte arrest at the metaphase I (MI) stage (control: 13.84 ± 0.95%; ßi: 31.30 ± 4.18%), which resulted in the reduction of the maturation rate (control: 80.36 ± 1.94%; ßi: 65.40 ± 1.14%). The level of phospho-GRK2 decreased in the treated group, suggesting that GRK2 activity was reduced upon GRK2 inhibition. Furthermore, the addition of ßi decreased Ca2+ release from the ER. The protein levels of cyclin B and cyclin-dependent kinase 1 were higher in the treatment group than those in the control group, indicating that GRK2 inhibition prevented a decrease in MPF activity. Collectively, GRK2 inhibition induced meiotic arrest at the MI stage in porcine oocytes by preventing a decrease in MPF activity, suggesting that GRK2 is essential for oocyte meiotic maturation in pigs.
Assuntos
Cálcio , Quinase 2 de Receptor Acoplado a Proteína G , Meiose , Oócitos , Animais , Oócitos/efeitos dos fármacos , Meiose/efeitos dos fármacos , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Feminino , Cálcio/metabolismo , Suínos , Fator Promotor de Maturação/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterináriaRESUMO
Gustavus, a positive regulator in arthropod reproduction, features a conserved SPRY and a C-terminal SOCS box domain and belongs to the SPSB protein family. The SPSB family, encompassing SPSB1 to SPSB4, plays pivotal roles in higher animals, including immune response, apoptosis, growth, and stress responses. In Neocaridina denticulata sinensis, alternative splicing yielded two NdGustavus isoforms, NdGusX1 and NdGusX2, with distinct expression patterns-high in ovaries and muscles, respectively, and across all ovarian germ cells. These isoforms showed similar expression dynamics during embryogenesis and significant upregulation post-copper ion exposure (P < 0.05). The in situ hybridization result elucidated that NdGusX1 and NdGusX2 were expressed across the germ cell spectrum in the ovary, with NdGusX1 showing enhanced expression in oogonia and primary oocytes. In addition, RNA interference revealed functional complementation in ovaries and potential functional differentiation in muscles. Knockdown of NdGusX1 and NdGusX2 potentially disrupted endogenous vitellogenin synthesis, regulating vitellogenesis and reducing mature oocyte volume, affecting follicular cavity occupation. This study provides a theoretical framework for understanding the biological functions of the SPSB family in crustacean ovarian maturation.
Assuntos
Processamento Alternativo , Ovário , Animais , Feminino , Ovário/metabolismo , Ovário/crescimento & desenvolvimento , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Oócitos/metabolismo , Vitelogênese/genética , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
A sex change phenomenon was reported in some free-living, non-sessile coral species of the Family Fungiidae. However, there are no reports describing sex change in sessile colonial species. Timing and cellular processes of sex change are also unclear in corals. Here, we report sex change of the colonial coral, Fimbriaphyllia ancora, and its cellular process. Of 26 colonies monitored at Nanwan Bay, southern Taiwan, about 70% changed their sex every year after annual spawning for least 3-4 consecutive years, i.e., colonies that were male two years ago became female last year, and male again this year. The remaining 30% were permanently male or female. Sex-change and non-sex-change colonies grew in close proximity or even side-by-side. No significant differences were found in colony size between sex-change and non-sex-change colonies. Histological analysis showed that, in female-to-male sex change, small oocytes were present up to 3 months in some gonads after spawning and disappeared by 5 months. This suggests that sex change occurred 4-5 months after spawning. In contrast, in male-to-female sex change, oocytes appeared weeks after sperm release and in most gonads by 3 months, suggesting that male-to-female sex change occurred 0-3 months after sperm release.
Assuntos
Antozoários , Reprodução , Animais , Antozoários/fisiologia , Antozoários/crescimento & desenvolvimento , Masculino , Feminino , Processos de Determinação Sexual , Taiwan , Gônadas/crescimento & desenvolvimento , OócitosRESUMO
BACKGROUND: With the trend toward late marriages and late childbearing, cryopreservation of oocytes for fertility preservation is attracting attention as a method to counteract the declining birthrate. OBJECTIVES: To examine the impact of social oocyte cryopreservation on local communities by assessing the significance of government assistance for cryofreezing and capturing the participants' subsequent feelings regarding this assistance. DESIGN: Descriptive study. METHODS: A prospective study was conducted on city-dwelling women <35 years old attending monthly seminars on oocyte retrieval/cryopreservation to whom the study concept was explained. Egg collection and storage management costs were free for 3 years after the project completed, and subsequent actual storage costs were borne by the individuals. After oocyte retrieval, we conducted a questionnaire on oocyte cryopreservation and administrative assistance. RESULTS: Of the 62 seminar participants, 2 became pregnant naturally without oocyte retrieval. Oocytes were retrieved in 34 women (average age: 32.8 years, number of oocytes obtained: 8.3), among whom 4 subsequently became pregnant and gave birth through natural pregnancy or artificial insemination, and 1 became pregnant and gave birth using frozen oocytes. In a follow-up questionnaire given to these 34 subjects, all responded that they were glad to have oocyte cryopreservation, but 23 subjects (67.6%) answered that they could not perform cryopreservation without financial assistance. Twenty-five participants (73.5%) wanted to try to conceive without using frozen oocytes as a post-cryopreservation plan. CONCLUSIONS: As a countermeasure against the declining birthrate, oocyte cryopreservation and associated workshops that can provide the information and education needed to conduct this task in a "planned" manner may be useful in providing women with additional reproductive options. Financial assistance will also be required to offer this service to the women who need it.
Women benefit when egg freezing is subsidized by local municipalitiesWhy was the study done? To prospectively examine the significance of egg freezing in a society in which the declining birthrate is an issue, particularly with regard to those who wish to undergo egg freezing and their trends when it is supported by the government. What did the researchers do? This project was conducted as a three-year endowed course by a local city government. Participants were women aged 20 to 34 who lived in the city and were recruited through the city's newsletter and website. They then attended a fertility workshop that was held once a month. Participants who wished to freeze their eggs were offered one free egg retrieval and three years of frozen storage. Participants were also asked to complete a questionnaire about their progress three years after the project ended. What did the researchers find? Sixty-two women participated in the three-year project, of whom 34 chose to freeze their eggs. Those who did not plan to conceive early, and two conceived naturally. Of those who froze their eggs, only one gave birth using the frozen eggs, and seven conceived naturally or through fertility treatments without using frozen eggs, two of whom had two pregnancies, resulting in 10 children being born. What do the findings mean? Three years after the project ended, the findings suggested that egg freezing itself may not have had a significant effect on pregnancy and childbirth but that holding workshops on fertility may have acted as an incentive for women to become pregnant and give birth.
Assuntos
Criopreservação , Preservação da Fertilidade , Oócitos , Humanos , Feminino , Criopreservação/métodos , Estudos Prospectivos , Adulto , Preservação da Fertilidade/métodos , Gravidez , Inquéritos e Questionários , Recuperação de OócitosRESUMO
Vitamin B12 is an essential nutritional co-factor for the folate and methionine cycles, which together constitute one-carbon metabolism. Here, we show that dietary uptake of vitamin B12 modulates cell fate decisions controlled by the conserved RAS/MAPK signaling pathway in C. elegans. A bacterial diet rich in vitamin B12 increases vulval induction, germ cell apoptosis and oocyte differentiation. These effects are mediated by different one-carbon metabolites in a tissue-specific manner. Vitamin B12 enhances via the choline/phosphatidylcholine metabolism vulval induction by down-regulating fat biosynthesis genes and increasing H3K4 tri-methylation, which results in increased expression of RAS/MAPK target genes. Furthermore, the nucleoside metabolism and H3K4 tri-methylation positively regulate germ cell apoptosis and oocyte production. Using mammalian cells carrying different activated KRAS and BRAF alleles, we show that the effects of methionine on RAS/MAPK-regulated phenotype are conserved in mammals. Our findings suggest that the vitamin B12-dependent one-carbon metabolism is a limiting factor for diverse RAS/MAPK-induced cellular responses.
Assuntos
Apoptose , Caenorhabditis elegans , Diferenciação Celular , Metionina , Vitamina B 12 , Animais , Vitamina B 12/metabolismo , Vitamina B 12/farmacologia , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Feminino , Metionina/metabolismo , Apoptose/efeitos dos fármacos , Oócitos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas ras/metabolismo , Carbono/metabolismo , Vulva/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células Germinativas/metabolismo , Colina/metabolismo , Fosfatidilcolinas/metabolismo , Camundongos , Humanos , Histonas/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: The aim of this study was to determine the effects of seasons (winter vs. summer) on oocyte quality in infertile women undergoing ovulation induction for in vitro fertilization. METHODS: This retrospective cross-sectional study assessed 155 cycles of in vitro fertilization-induced ovulation in women, with 71 and 84 cycles occurring in the summer and winter, respectively. Oocytes were evaluated for quality, with 788 and 713 assessed during summer and winter, and classified according to Nikiforov's categories: (a) category I, good quality; (b) category 2, medium quality; and (c) category 3, low quality. RESULTS: Thickened zona pellucida (p<0.001), increased perivitelline space (p<0.001), oocyte shape abnormalities (p=0.01), and the presence of refractile bodies (p<0.0001) were more frequent in the summer cycles, whereas cytoplasmic granularity (p<0.001) was more frequent in the winter cycles. In winter, we observed a higher frequency of category 3 (p<0.001) and category 2 (p<0.001) oocytes and a lower frequency of category 1 (p<0.001) oocytes. CONCLUSION: Oocyte dysmorphisms were found in 70-80% of cases and were more common in winter. The main features include a thickened zona pellucida, enlarged perivitelline space, irregular shape, and cytoplasmic granularity. This implies better-quality oocytes in the summer than in the winter. However, retrospective studies have limitations due to data collection biases and potential confounding variables such as diet and exercise. Future research is needed to confirm these findings and explore the underlying mechanisms.
Assuntos
Fertilização in vitro , Oócitos , Estações do Ano , Humanos , Feminino , Estudos Retrospectivos , Estudos Transversais , Oócitos/fisiologia , Adulto , Infertilidade Feminina/terapia , Indução da OvulaçãoRESUMO
In-vitro maturation (IVM) is the process of cultivating early-stage follicles from the primordial to the antral stage and facilitating the maturation of oocytes outside the body within a supportive environment. This intricate procedure requires the careful coordination of various factors to replicate the natural ovarian conditions. Advanced techniques for IVM are designed to mimic the natural ovarian environment and enhance the development of follicles. Three-dimensional (3D) culture systems provide a more biologically relevant setting for follicle growth compared to traditional two-dimensional (2D) cultures. Traditional culture systems, often fail to support the complex process of follicle development effectively. However, modern engineered reproductive tissues and culture systems are making it possible to create increasingly physiological in-vitro models of folliculogenesis. These innovative methods are enabling researchers and clinicians to better replicate the dynamic and supportive environment of the ovary, thereby improving the outcomes of IVM offering new hope for fertility preservation and treatment. This paper focuses on the routine 3D culture, and innovative 3D culture of ovary and follicles, including a tissue engineering scaffolds, microfluidic (dynamic) culture system, organ-on-chip models, EVATAR system, from a clinical perspective to determine the most effective approach for achieving in-vitro maturation of follicles. These techniques provide critical support for ovarian function in various ovarian-associated disorders, including primary ovarian insufficiency (POI), premature ovarian failure (POF), ovarian cancer, and age-related infertility.
Assuntos
Preservação da Fertilidade , Folículo Ovariano , Engenharia Tecidual , Feminino , Humanos , Engenharia Tecidual/métodos , Preservação da Fertilidade/métodos , Ovário/fisiologia , Animais , Oócitos/fisiologia , Alicerces Teciduais , Técnicas de Cultura de Células em Três Dimensões/métodos , Insuficiência Ovariana Primária/terapiaRESUMO
BACKGROUND: Controlled ovarian stimulation is a common skill of assisted reproductive technologies (ARTs). In the clinic, some females would undergo more than one controlled ovarian stimulation cycle. However, few studies have focused on the influence of multi-superovulation on oocytes and offspring. RESULTS: Here, we found that multi-superovulation disrupted the transcriptome of oocytes and that the differentially expressed genes (DEGs) were associated mainly with metabolism and fertilization. The disruption of mRNA degradation via poly (A) size and metabolism might be a reason for the reduced oocyte maturation rate induced by repeated superovulation. Multi-superovulation results in hypo-genomic methylation in oocytes. However, there was an increase in the methylation level of CGIs. The DMRs are not randomly distributed in genome elements. Genes with differentially methylated regions (DMRs) in promoters are enriched in metabolic pathways. With increasing of superovulation cycles, the glucose and insulin tolerance of offspring is also disturbed. CONCLUSIONS: These results suggest that multi-superovulation has adverse effects on oocyte quality and offspring health.
Assuntos
Metilação de DNA , Oócitos , Superovulação , Oócitos/metabolismo , Metilação de DNA/genética , Feminino , Superovulação/genética , Superovulação/efeitos dos fármacos , Animais , Humanos , Transcriptoma/genética , Camundongos , Indução da Ovulação/métodos , Ilhas de CpG/genéticaRESUMO
The field of reproductive biology has made significant progress in recent years, identifying specific molecular players that influence oocyte development and function. Among them, sirtuin 3 (SIRT3) has attracted particular attention for its central role in mediating mitochondrial function and cellular stress responses in oocytes. So far, studies have demonstrated that the knockdown of SIRT3 leads to a decrease in blastocyst formation and an increase in oxidative stress within an embryo, underscoring the importance of SIRT3 in maintaining the cellular redox balance critical for embryonic survival and growth. Furthermore, the literature reveals specific signaling pathways, such as the SIRT3- Glycogen synthase kinase-3 beta (GSK3ß) deacetylation pathway, crucial for mitigating oxidative stress-related anomalies in oocyte meiosis, particularly under conditions like maternal diabetes. Overall, the emerging role of SIRT3 in regulating oocyte mitochondrial function and development highlights the critical importance of understanding the intricate connections between cellular metabolism, stress response pathways, and overall reproductive health and function. This knowledge could lead to the development of novel strategies to support oocyte quality and fertility, with far-reaching implications for assisted reproductive technologies and women's healthcare. This commentary aims to provide an overview of the importance of SIRT3 in oocytes by synthesizing results from a multitude of studies. The aim is to elucidate the role of SIRT3 in oocyte development, maturation, and aging and to identify areas where further research is needed.
Assuntos
Oócitos , Sirtuína 3 , Oócitos/metabolismo , Sirtuína 3/metabolismo , Sirtuína 3/genética , Humanos , Animais , Mitocôndrias/metabolismo , Senescência Celular , Feminino , Mamíferos/metabolismo , Estresse OxidativoRESUMO
The insecticidal crystalline (Cry) and vegetative insecticidal (Vip) proteins derived from Bacillus thuringiensis (Bt) are used globally to manage insect pests, including the cotton bollworm, Helicoverpa armigera, one of the world's most damaging agricultural pests. Cry proteins bind to the ATP-binding cassette transporter C2 (ABCC2) receptor on the membrane surface of larval midgut cells, resulting in Cry toxin pores, and ultimately leading to cell swelling and/or lysis. Insect aquaporin (AQP) proteins within the membranes of larval midgut cells are proposed to allow the rapid influx of water into enterocytes following the osmotic imbalance triggered by the formation of Cry toxin pores. Here, we examined the involvement of H. armigera AQPs in Cry1Ac-induced osmotic cell swelling. We identified and characterized eight H. armigera AQPs and demonstrated that five are functional water channel proteins. Three of these (HaDrip1, HaPrip, and HaEglp1) were found to be expressed in the larval midgut. Xenopus laevis oocytes co-expressing the known Cry1Ac receptor HaABCC2 and each of the three HaAQPs displayed abnormal morphology and were lysed following exposure to Cry1Ac, suggesting a rapid influx of water was induced after Cry1Ac pore formation. In contrast, oocytes producing either HaABCC2 or HaAQP alone failed to swell or lyse after treatment with Cry1Ac, implying that both Cry1Ac pore formation and HaAQP function are needed for osmotic cell swelling. However, CRISPR/Cas9-mediated knockout of any one of the three HaAQP genes failed to cause significant changes in susceptibility to the Bt toxins Cry1Ac, Cry2Ab, or Vip3Aa. Our findings suggest that the multiple HaAQPs produced in larval midgut cells compensate for each other in allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation, and that mutations affecting a single HaAQP are unlikely to confer resistance to Bt proteins.
Assuntos
Aquaporinas , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas , Proteínas Hemolisinas , Larva , Mariposas , Animais , Toxinas de Bacillus thuringiensis/toxicidade , Proteínas Hemolisinas/toxicidade , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/metabolismo , Endotoxinas/toxicidade , Endotoxinas/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Mariposas/efeitos dos fármacos , Mariposas/metabolismo , Mariposas/genética , Larva/efeitos dos fármacos , Larva/metabolismo , Aquaporinas/metabolismo , Aquaporinas/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/genética , Xenopus laevis , Oócitos/metabolismo , Oócitos/efeitos dos fármacos , Inseticidas/toxicidade , Inseticidas/farmacologia , Osmose , Helicoverpa armigeraRESUMO
Oogenesis involves two phases: initial volumetric growth driven by nutrient accumulation and subsequent nuclear maturation. While melatonin (MLT) has been employed as a supplement to enhance the quality of fully grown oocytes during nuclear maturation phase, its impact on oocyte growth remains poorly studied. Here, we provide in vivo evidence demonstrating that follicle-stimulating hormone increases MLT content in ovary. Administration of MLT improves oocyte growth and quality in mice and goats by enhancing nutrient reserves and mitochondrial function. Conversely, MLT-deficient mice have smaller oocytes and dysfunctional mitochondria. Exploring the clinical implications of MLT in promoting oocyte growth, we observe that a brief 2-day MLT treatment enhances oocyte quality and reproductive performance in older mice. These findings highlight the role of MLT in regulating oocyte growth and provide a specific treatment window for optimizing oocyte quality and reproductive performance in female animals.
Assuntos
Cabras , Melatonina , Mitocôndrias , Oócitos , Animais , Melatonina/farmacologia , Melatonina/metabolismo , Oócitos/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Camundongos , Feminino , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Hormônio Foliculoestimulante/metabolismo , Nutrientes/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Reproductive aging is a major cause of fertility decline, attributed to decreased oocyte quantity and developmental potential. A possible cause is aging of the surrounding follicular somatic cells that support oocyte growth and development by providing nutrients and regulatory factors. Here, by creating chimeric follicles, whereby an oocyte from one follicle was transplanted into and cultured within another follicle whose native oocyte was removed, we show that young oocytes cultured in aged follicles exhibited impeded meiotic maturation and developmental potential, whereas aged oocytes cultured within young follicles were significantly improved in rates of maturation, blastocyst formation and live birth after in vitro fertilization and embryo implantation. This rejuvenation of aged oocytes was associated with enhanced interaction with somatic cells, transcriptomic and metabolomic remodeling, improved mitochondrial function and higher fidelity of meiotic chromosome segregation. These findings provide the basis for a future follicular somatic cell-based therapy to treat female infertility.
Assuntos
Oócitos , Folículo Ovariano , Rejuvenescimento , Feminino , Animais , Folículo Ovariano/crescimento & desenvolvimento , Rejuvenescimento/fisiologia , Camundongos , Fertilização in vitro/métodos , Senescência Celular , Meiose , Microambiente Celular , Envelhecimento/fisiologiaRESUMO
The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles.
Assuntos
Trifosfato de Adenosina , Toxinas Bacterianas , Cálcio , Canais de Cloreto , Vesículas Extracelulares , Oócitos , Xenopus laevis , Animais , Oócitos/metabolismo , Oócitos/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Canais de Cloreto/metabolismo , Humanos , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Feminino , Clostridium perfringens/metabolismoRESUMO
During eukaryotic cell division, a microtubule-based structure called the spindle exerts forces on chromosomes. The best-studied spindle forces, including those responsible for the separation of sister chromatids, are directed parallel to the spindle's long axis. By contrast, little is known about forces perpendicular to the spindle axis, which determine the metaphase plate configuration and thus the location of chromosomes in the subsequent nucleus. Using live-cell microscopy, we find that metaphase chromosomes are spatially anti-correlated in mouse oocyte spindles, evidence of previously unknown long-range forces acting perpendicular to the spindle axis. We explain this observation by showing that the spindle's microtubule network behaves as a nematic liquid crystal and that deformation of the nematic field around embedded chromosomes causes long-range repulsion between them.
Assuntos
Microtúbulos , Oócitos , Fuso Acromático , Animais , Fuso Acromático/metabolismo , Oócitos/metabolismo , Oócitos/citologia , Camundongos , Microtúbulos/metabolismo , Metáfase , Cromossomos , Cromossomos de Mamíferos/metabolismo , FemininoRESUMO
Assisted reproductive technologies are an emerging field in equine reproduction, with species-dependent peculiarities, such as the low success rate of conventional IVF. Here, the 'cumulome' was related to the developmental capacity of its corresponding oocyte. Cumulus-oocyte complexes collected from slaughterhouse ovaries were individually matured, fertilized by ICSI, and cultured. After maturation, the cumulus was collected for proteomics analysis using label-free mass spectrometry (MS)-based protein profiling by nano-HPLC MS/MS and metabolomics analysis by UPLC-nanoESI MS. Overall, a total of 1671 proteins and 612 metabolites were included in the quantifiable 'cumulome'. According to the development of the corresponding oocytes, three groups were compared with each other: not matured (NM; n = 18), cleaved (CV; n = 15), and blastocyst (BL; n = 19). CV and BL were also analyzed together as the matured group (M; n = 34). The dataset revealed a closer connection within the two M groups and a more distinct separation from the NM group. Overrepresentation analysis detected enrichments related to energy metabolism as well as vesicular transport in the M group. Functional enrichment analysis found only the KEGG pathway 'oxidative phosphorylation' as significantly enriched in the NM group. A compound attributed to ATP was observed with significantly higher concentrations in the BL group compared with the NM group. Finally, in the NM group, proteins related to degradation of glycosaminoglycans were lower and components of cumulus extracellular matrix were higher compared to the other groups. In summary, the study revealed novel pathways associated with the maturational and developmental competence of oocytes.
Assuntos
Células do Cúmulo , Técnicas de Maturação in Vitro de Oócitos , Oócitos , Animais , Cavalos , Oócitos/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/citologia , Feminino , Células do Cúmulo/metabolismo , Proteômica/métodos , Blastocisto/metabolismo , Blastocisto/citologia , Metabolômica/métodos , Espectrometria de Massas em Tandem , Injeções de Esperma IntracitoplásmicasRESUMO
Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.