RESUMO
Spindle bipolarization, the process of a microtubule mass transforming into a bipolar spindle, is a prerequisite for accurate chromosome segregation. In contrast to mitotic cells, the process and mechanism of spindle bipolarization in human oocytes remains unclear. Using high-resolution imaging in more than 1800 human oocytes, we revealed a typical state of multipolar intermediates that form during spindle bipolarization and elucidated the mechanism underlying this process. We found that the minor poles formed in multiple kinetochore clusters contribute to the generation of multipolar intermediates. We further determined the essential roles of HAUS6, KIF11, and KIF18A in spindle bipolarization and identified mutations in these genes in infertile patients characterized by oocyte or embryo defects. These results provide insights into the physiological and pathological mechanisms of spindle bipolarization in human oocytes.
Assuntos
Segregação de Cromossomos , Cinesinas , Cinetocoros , Microtúbulos , Oócitos , Fuso Acromático , Humanos , Oócitos/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Cinetocoros/metabolismo , Fuso Acromático/metabolismo , Microtúbulos/metabolismo , Feminino , Mutação , Polos do Fuso/metabolismoAssuntos
Meiose , Oócitos , Humanos , Oócitos/crescimento & desenvolvimento , Meiose/genética , FemininoRESUMO
The objective of this study was to evaluate clinical outcomes for patients undergoing IVF treatment where an artificial intelligence (AI) platform was utilized by clinicians to help determine the optimal starting dose of FSH and timing of trigger injection. This was a prospective clinical trial with historical control arm. Four physicians from two assisted reproductive technology treatment centers in the United States participated in the study. The treatment arm included patients undergoing autologous IVF cycles between December 2022-April 2023 where the physician use AI to help select starting dose of follicle stimulating hormone (FSH) and trigger injection timing (N = 291). The control arm included historical patients treated where the same doctor did not use AI between September 2021 and September 2022. The main outcome measures were total FSH used and average number of mature metaphase II (MII) oocytes. There was a non-significant trend towards improved patient outcomes and a reduction in FSH with physician use of AI. Overall, the average number of MIIs in the treatment vs. control arm was 12.20 vs 11.24 (improvement = 0.96, p = 0.16). The average number of oocytes retrieved in the treatment vs. control arm was 16.01 vs 14.54 (improvement = 1.47, p = 0.08). The average total FSH in the treatment arm was 3671.95 IUs and the average in the control arm was 3846.29 IUs (difference = -174.35 IUs, p = 0.13). These results suggests that AI can safely assist in refining the starting dose of FSH while narrowing down the timing of the trigger injection during ovarian stimulation, benefiting the patient in optimizing the count of MII oocytes retrieved.
Assuntos
Fertilização in vitro , Hormônio Foliculoestimulante , Aprendizado de Máquina , Oócitos , Indução da Ovulação , Humanos , Feminino , Estudos Prospectivos , Adulto , Hormônio Foliculoestimulante/administração & dosagem , Indução da Ovulação/métodos , Fertilização in vitro/métodos , Oócitos/citologia , Recuperação de Oócitos/métodos , GravidezRESUMO
BACKGROUND: Obesity is a global health issue with detrimental effects on various human organs, including the reproductive system. Observational human data and several lines of animal experimental data suggest that maternal obesity impairs ovarian function and early embryo development, but the precise pathogenesis remains unclear. METHODS: We established a high-fat diet (HFD)-induced obese female mouse model to assess systemic metabolism, ovarian morphology, and oocyte function in mice. For the first time, this study employed single-cell RNA sequencing to explore the altered transcriptomic landscape of preimplantation embryos at different stages in HFD-induced obese mice. Differential gene expression analysis, enrichment analysis and protein-protein interactions network analysis were performed. RESULTS: HFD-induced obese female mice exhibited impaired glucolipid metabolism and insulin resistance. The ovaries of HFD mice had a reduced total follicle number, an increased proportion of atretic follicles, and irregular granulosa cell arrangement. Furthermore, the maturation rate of embryonic development by in vitro fertilization of oocytes was significantly decreased in HFD mice. Additionally, the transcriptional landscapes of preimplantation embryos at different stages in mice induced by different diets were significantly distinguished. The maternal-to-zygotic transition was also affected by the failure to remove maternal RNAs and to turn off zygotic genome expression. CONCLUSIONS: HFD-induced obesity impaired ovarian morphology and oocyte function in female mice and further led to alterations in the transcriptional landscape of preimplantation embryos at different stages of HFD mice.
Assuntos
Dieta Hiperlipídica , Desenvolvimento Embrionário , Obesidade , Oócitos , Análise de Sequência de RNA , Análise de Célula Única , Animais , Feminino , Dieta Hiperlipídica/efeitos adversos , Oócitos/metabolismo , Camundongos , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Obesidade/genética , Obesidade/metabolismo , Camundongos Endogâmicos C57BL , Gravidez , Blastocisto/metabolismoRESUMO
OBJECTIVE: The aim of this study was to compare the obstetric and perinatal complications in women who became pregnant with autologous oocytes and those who received donated oocytes (DO) in intracytoplasmic sperm injection cycles (ICSI). METHODS: A retrospective cohort study was carried out by collecting data from medical records between 2019 and 2022. Only patients who underwent ICSI in an induced cycle using their own or freshly DO, with male infertility factor and tubal factor, were included. RESULTS: A total of 120 patients were assessed, comprising 51 cases utilizing their own oocytes (control group) and 69 cases employing DO (study group). Patients receiving DO (n=69) exhibited a significantly higher mean age compared to those utilizing their own oocytes (n=51) (41.96±2.16 vs 38.54±1.42 years, p<0.001). There was no significant association between the source of oocytes and gestational age at delivery (p=0.296), birth weight (p=0.836), admission to neonatal intensive care unit (ICU) (p=0.120), or maternal admission to adult ICU (p=0.767). Additionally, the origin of oocytes did not demonstrate any significant association with the risk of pre-eclampsia (p=0.357), gestational diabetes mellitus (p=0.187), premature rupture of membranes (p=0.996), uterine atony (p=0.996), placenta previa (p=0.393), oligohydramnios (p=0.393), or gestational hypertension (p=0.393)." CONCLUSION: An increase in obstetric and perinatal complications was not observed in pregnancies with DO compared to pregnancies with autologous oocytes in women undergoing ICSI without prior comorbidities. Further studies with larger sample sizes are required to validate our findings.
Assuntos
Doação de Oócitos , Injeções de Esperma Intracitoplásmicas , Humanos , Injeções de Esperma Intracitoplásmicas/efeitos adversos , Feminino , Gravidez , Estudos Retrospectivos , Adulto , Doação de Oócitos/efeitos adversos , Recém-Nascido , Complicações na Gravidez , Masculino , Resultado da Gravidez , Oócitos , Fatores de RiscoRESUMO
Advanced glycation end products (AGEs) are the final products of the Maillard reaction, formed through the interaction of carbohydrates and proteins. Reactive dicarbonyl compounds such as methylglyoxal (MGO) serve as precursors for AGEs formation. Elevated levels of MGO/AGEs are observed in conditions like obesity, polycystic ovarian syndrome (PCOS), and diabetes, negatively impacting oocyte development. Previous studies have shown that hydrogen sulfide, a gasotransmitter with anti-AGEs effects, is produced in a process influenced by vitamin B6. R-α-lipoic acid (ALA) inhibits protein glycation and AGEs formation while stimulating glutathione (GSH) production. Taurine mitigates oxidative stress and acts as an anti-glycation compound, preventing in vitro glycation and AGEs accumulation. This study aimed to explore the ameliorative effects of a micronutrient support (Taurine, ALA and B6: TAB) on mouse oocytes challenged with MGO. Our results indicate that MGO reduces oocyte developmental competence, while TAB supplementation improves maturation, fertilization, and blastocyst formation rates. TAB also restores cell lineage allocation, redox balance and mitigates mitochondrial dysfunction in MGO-challenged oocytes. Furthermore, cumulus cells express key enzymes in the transsulfuration pathway, and TAB enhances their mRNA expression. However, TAB does not rescue MGO-induced damage in denuded oocytes, emphasizing the supportive role of cumulus cells. Overall, these findings suggest that TAB interventions may have significant implications for addressing reproductive dysfunctions associated with elevated MGO/AGEs levels. This study highlights the potential of TAB supplementation in preserving the developmental competence of COCs exposed to MGO stress, providing insights into mitigating the impact of dicarbonyl stress on oocyte quality and reproductive outcomes.
Assuntos
Oócitos , Aldeído Pirúvico , Taurina , Ácido Tióctico , Vitamina B 6 , Animais , Taurina/farmacologia , Aldeído Pirúvico/farmacologia , Aldeído Pirúvico/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Camundongos , Ácido Tióctico/farmacologia , Feminino , Vitamina B 6/farmacologia , Vitamina B 6/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacosRESUMO
DNA damage is a key factor affecting gametogenesis and embryo development. The integrity and stability of DNA are fundamental to a woman's successful conception, embryonic development, pregnancy and the production of healthy offspring. Aging, reactive oxygen species, radiation therapy, and chemotherapy often induce oocyte DNA damage, diminished ovarian reserve, and infertility in women. With the increase of infertility population, there is an increasing need to study the relationship between infertility related diseases and DNA damage and repair. Researchers have tried various methods to reduce DNA damage in oocytes and enhance their DNA repair capabilities in an attempt to protect oocytes. In this review, we summarize recent advances in the DNA damage response mechanisms in infertility diseases such as PCOS, endometriosis, diminished ovarian reserve and hydrosalpinx, which has important implications for fertility preservation.
Assuntos
Dano ao DNA , Reparo do DNA , Infertilidade Feminina , Feminino , Humanos , Infertilidade Feminina/genética , Infertilidade Feminina/terapia , Oócitos , Síndrome do Ovário Policístico/genética , Endometriose/genética , Reserva Ovariana/fisiologia , Preservação da Fertilidade/métodosRESUMO
Mutations in SCN4A gene encoding Nav1.4 channel α-subunit, are known to cause neuromuscular disorders such as myotonia or paralysis. Here, we study the effect of two amino acid replacements, K1302Q and G1306E, in the DIII-IV loop of the channel, corresponding to mutations found in patients with myotonia. We combine clinical, electrophysiological, and molecular modeling data to provide a holistic picture of the molecular mechanisms operating in mutant channels and eventually leading to pathology. We analyze the existing clinical data for patients with the K1302Q substitution, which was reported for adults with or without myotonia phenotypes, and report two new unrelated patients with the G1306E substitution, who presented with severe neonatal episodic laryngospasm and childhood-onset myotonia. We provide a functional analysis of the mutant channels by expressing Nav1.4 α-subunit in Xenopus oocytes in combination with ß1 subunit and recording sodium currents using two-electrode voltage clamp. The K1302Q variant exhibits abnormal voltage dependence of steady-state fast inactivation, being the likely cause of pathology. K1302Q does not lead to decelerated fast inactivation, unlike several other myotonic mutations such as G1306E. For both mutants, we observe increased window currents corresponding to a larger population of channels available for activation. To elaborate the structural rationale for our experimental data, we explore the contacts involving K/Q1302 and E1306 in the AlphaFold2 model of wild-type Nav1.4 and Monte Carlo-minimized models of mutant channels. Our data provide the missing evidence to support the classification of K1302Q variant as likely pathogenic and may be used by clinicians.
Assuntos
Miotonia , Canal de Sódio Disparado por Voltagem NAV1.4 , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Humanos , Animais , Miotonia/genética , Feminino , Xenopus laevis , Masculino , Mutação , Oócitos/metabolismo , Adulto , Substituição de AminoácidosRESUMO
Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. Biochemical characterization was conducted using the Xenopus laevis oocyte expression system to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, translocating 1 mM of substrate within 25 min. The transporter displayed strict directionality and specificity for secologanin and did not accept other secoiridoid substrates. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of pathway flux, mediating the balance between a defense arsenal and cellular homeostasis.
Assuntos
Catharanthus , Proteínas de Plantas , Alcaloides de Triptamina e Secologanina , Vacúolos , Catharanthus/metabolismo , Catharanthus/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Vacúolos/metabolismo , Alcaloides de Triptamina e Secologanina/metabolismo , Animais , Filogenia , Xenopus laevis/metabolismo , Transporte Biológico , Oócitos/metabolismo , Glucosídeos IridoidesRESUMO
BACKGROUND: Appropriate regulation of genes expressed in oocytes and embryos is essential for acquisition of developmental competence in mammals. Here, we hypothesized that several genes expressed in oocytes and pre-implantation embryos remain unknown. Our goal was to reconstruct the transcriptome of oocytes (germinal vesicle and metaphase II) and pre-implantation cattle embryos (blastocysts) using short-read and long-read sequences to identify putative new genes. RESULTS: We identified 274,342 transcript sequences and 3,033 of those loci do not match a gene present in official annotations and thus are potential new genes. Notably, 63.67% (1,931/3,033) of potential novel genes exhibited coding potential. Also noteworthy, 97.92% of the putative novel genes overlapped annotation with transposable elements. Comparative analysis of transcript abundance identified that 1,840 novel genes (recently added to the annotation) or potential new genes were differentially expressed between developmental stages (FDR < 0.01). We also determined that 522 novel or potential new genes (448 and 34, respectively) were upregulated at eight-cell embryos compared to oocytes (FDR < 0.01). In eight-cell embryos, 102 novel or putative new genes were co-expressed (|r|> 0.85, P < 1 × 10-8) with several genes annotated with gene ontology biological processes related to pluripotency maintenance and embryo development. CRISPR-Cas9 genome editing confirmed that the disruption of one of the novel genes highly expressed in eight-cell embryos reduced blastocyst development (ENSBTAG00000068261, P = 1.55 × 10-7). CONCLUSIONS: Our results revealed several putative new genes that need careful annotation. Many of the putative new genes have dynamic regulation during pre-implantation development and are important components of gene regulatory networks involved in pluripotency and blastocyst formation.
Assuntos
Blastocisto , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Oócitos , Animais , Bovinos , Desenvolvimento Embrionário/genética , Oócitos/metabolismo , Blastocisto/metabolismo , Transcriptoma , Anotação de Sequência Molecular , Perfilação da Expressão Gênica , FemininoRESUMO
Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming was examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions-consistent with crystallization of most free water-during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-warming oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.
Assuntos
Criopreservação , Crioprotetores , Gelo , Oócitos , Criopreservação/métodos , Animais , Crioprotetores/farmacologia , Bovinos , Feminino , Difração de Raios XRESUMO
The ovarian cortical reserve of follicles is vital for fertility. Some medical treatments are toxic to follicles, leading to premature ovarian insufficiency. Ovarian tissue cryopreservation is an established method to preserve fertility in adults and even applied in prepuberty despite unproven efficacy. Here, we analyze transcriptomes of 120 cortical follicles from children and adults for detailed comparison. We discover heterogeneity with two main types of follicles in both age groups: one with expected oocyte-granulosa profiles and another with predicted role in signaling. Transcriptional changes during growth to the secondary stage are similar overall in children and adults, but variations related to extracellular matrix, theca cells, and miRNA profiles are found. Notably, cyclophosphamide dose correlates with interferon signaling in child follicles. Additionally, morphology alone is insufficient for follicle categorization suggesting a need for additional markers. Marker genes for early follicle activation are determined. These findings will help refine follicular classification and fertility preservation techniques across critical ages.
Assuntos
Folículo Ovariano , Transcriptoma , Feminino , Humanos , Folículo Ovariano/metabolismo , Adulto , Criança , Oócitos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Perfilação da Expressão Gênica , Ciclofosfamida , Criopreservação , Preservação da Fertilidade/métodos , Adolescente , Transdução de Sinais , Pré-EscolarRESUMO
This study aimed to evaluate the effect of chemical gasification and HEPES as alternative systems to pH control during in vitro maturation on bovine oocytes competence. Groups of 20 bovine cumulus oocytes complexes (COCs) were randomly distributed and cultured for 24 h in one of the following experimental groups: (i) chemical reaction (ChRG) system: CO2 generated from sodium bicarbonate and citric acid reaction (ii) culture media TCM-HEPES (HEPES-G); and (iii) control group (CNTG) in conventional incubator. After in vitro maturation (IVM), the COCs were in vitro fertilized (IVF), and in vitro cultivated (IVC) in a conventional incubator. We evaluated oocyte nuclear maturation, cleavage and blastocyst rates, in addition to the relative mRNA expression of BAX, BMP-15, AREG and EREG genes in oocytes and cumulus cells. The proportion of oocytes in metaphase II was higher in CNTG and ChRG (77.57% and 77.06%) than in the HEPES-G (65.32%; p = .0408 and .0492, respectively). The blastocyst production was similar between CNTG and ChRG (26.20% and 28.47%; p = .4232) and lower (p = .001) in the HEPES-G (18.71%). The relative mRNA expression of BAX gene in cumulus cells was significantly higher (p = .0190) in the HEPES-G compared to the CNTG. Additionally, the relative mRNA expression of BMP-15 gene was lower (p = .03) in oocytes from HEPES-G compared to the CNTG. In conclusion, inadequate atmosphere control has a detrimental effect on oocyte maturation. Yet, the use of chemical gasification can be an efficient alternative to bovine COCs cultivation.
Assuntos
Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos , Oócitos , Animais , Bovinos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/efeitos dos fármacos , Fertilização in vitro/veterinária , Feminino , Meios de Cultura , Blastocisto/efeitos dos fármacos , Células do Cúmulo/efeitos dos fármacos , Dióxido de Carbono/farmacologia , Bicarbonato de Sódio/farmacologia , Ácido Cítrico/farmacologia , Técnicas de Cultura Embrionária/veterináriaRESUMO
The study of oocyte development holds significant implications in developmental biology. The zebrafish (Danio rerio) has been extensively used as a model organism to investigate early developmental processes from oocyte to embryo. In zebrafish, oocytes are surrounded by a single layer of somatic granulosa cells. However, separating granulosa cells from oocytes poses a challenge, as achieving pure oocytes is crucial for precise analysis. Although various methods have been proposed to isolate zebrafish oocytes at different developmental stages, current techniques fall short in removing granulosa cells completely, limiting the accuracy of genome analysis focused solely on oocytes. In this study, we successfully developed a rapid and efficient process for isolating pure stage I oocytes in zebrafish while eliminating granulosa cell contamination. This technique facilitates biochemical and molecular analysis, particularly in exploring epigenetic and genome structure aspects specific to oocytes. Notably, the method is user-friendly, minimizes oocyte damage, and provides a practical solution for subsequent research and analysis.
Assuntos
Células da Granulosa , Oócitos , Peixe-Zebra , Animais , Oócitos/citologia , Feminino , Células da Granulosa/citologiaRESUMO
This study used the brilliant cresyl blue (BCB) staining method to group buffalo oocytes (BCB+ and BCB-) and perform in vitro maturation, in vitro fertilization and embryo culture. At the same time, molecular biology techniques were used to detect gap junction protein expression and oxidative stress-related indicators to explore the molecular mechanism of BCB staining to predict oocyte developmental potential. The techniques of buffalo oocytes to analyse their developmental potential and used immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels and qPCR to detect the expression levels of the antioxidant-related genes SOD2 and GPX1. Our results showed that the in vitro maturation rate, embryo cleavage rate and blastocyst rate of buffalo oocytes in the BCB+ group were significantly higher than those in the BCB- group and the control group (p < .05). The expression level of CX43 protein in the BCB+ group was higher than that in the BCB- group both before and after maturation (p < .05). The intensity of ROS in the BCB+ group was significantly lower than that in the BCB- group (p < .05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB+ group were significantly higher than those in the BCB- group (p < .05). Brilliant cresyl blue staining could effectively predict the developmental potential of buffalo oocytes. The results of BCB staining were positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the reactive oxygen species level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes might be closely related to antioxidant activity.
Assuntos
Búfalos , Conexina 43 , Técnicas de Maturação in Vitro de Oócitos , Oócitos , Oxazinas , Estresse Oxidativo , Animais , Oócitos/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterinária , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase/genética , Glutationa Peroxidase/metabolismo , Glutationa Peroxidase/genética , Fertilização in vitro/veterinária , Técnicas de Cultura Embrionária/veterinária , Glutationa Peroxidase GPX1 , Desenvolvimento Embrionário/fisiologia , Coloração e Rotulagem , Antioxidantes/metabolismoRESUMO
During maturation oocytes at the germinal vesicle (GV) stage progress to metaphase II (MII). However, during in vitro maturation a proportion often fail to progress. To understand these processes, we employed RNA sequencing to examine the transcriptome profile of these three groups of oocytes from the pig. We compared our findings with similar public oocyte data from humans. The transcriptomes in oocytes that failed to progress was similar to those that did. We found in both species, the most upregulated genes in MII oocytes were associated with chromosome segregation and cell cycle processes, while the most down regulated genes were relevant to ribosomal and mitochondrial pathways. Moreover, those genes involved in chromosome segregation during GV to MII transition were conserved in pig and human. We also compared MII and GV oocyte transcriptomes at the isoform transcript level in both species. Several thousands of genes (including DTNBP1, MAPK1, RAB35, GOLGA7, ATP1A1 and ATP2B1) identified as not different in expression at a gene transcript level were found to have differences in isoform transcript levels. Many of these genes were involved in ATPase-dependent or GTPase-dependent intracellular transport in pig and human, respectively. In conclusion, our study suggests the failure to progress to MII in vitro may not be regulated at the level of the genome and that many genes are differentially regulated at the isoform level, particular those involved ATPase- or GTPase-dependent intracellular transport.
Assuntos
Metáfase , Oócitos , Humanos , Oócitos/metabolismo , Oócitos/citologia , Animais , Suínos , Feminino , Transcriptoma , Análise de Sequência de RNA , Oogênese/genética , Perfilação da Expressão GênicaRESUMO
BACKGROUND: Vitrification is commonly used for in vitro fertilization and has significant impact on gametes. OBJECTIVE: To investigate changes in ultrastructure, membrane potential and distribution of mitochondria in mouse oocytes after vitriï¬cation. MATERIALS AND METHODS: Mouse oocytes were divided into three groups: one group as fresh control, one group for the toxicity test (treated with cryoprotectant but without vitrification), and the other for vitrification. RESULTS: Most mitochondria in oocytes were damaged after cooling and warming, being rough and fuzzy in appearance, even swollen and broken. The membrane potential of the toxicity test group and the vitrification group was 0.320 +/-0.030 and 0.244 +/- 0.038, respectively, in comparison to the fresh group (0.398 +/- 0.043). The membrane potential of the vitrified oocytes was significantly lower than fresh oocytes and the toxicity test oocytes (P % 0.05), but there was no significant difference between fresh oocytes and the toxicity test oocytes (P > 0.05). Mitochondria in fresh oocytes were denser and strained stronger, with 59.5> distributed homogeneously and 36.4> polarized. The majority of mitochondria in the toxicity-tested oocytes were clustered (69.3>) and only a small portion were distributed homogeneously (19.6>), while mitochondria in vitrified oocytes were clustered (56.3>) and deficient (24.4>), and their fluorescent staining was weak and blurred. There was a significant disruption in mitochondrial function after vitrification. CONCLUSION: Vitrification alters the ultrastructure, membrane potential and distribution of mitochondria in oocytes, most likely caused by toxicity and mechanical injury. Doi.org/10.54680/fr24510110212.
Assuntos
Criopreservação , Crioprotetores , Potencial da Membrana Mitocondrial , Mitocôndrias , Oócitos , Vitrificação , Animais , Oócitos/efeitos dos fármacos , Oócitos/ultraestrutura , Oócitos/citologia , Camundongos , Criopreservação/métodos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Mitocôndrias/metabolismo , Feminino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Crioprotetores/farmacologiaRESUMO
Oogenesis is the central process required to produce viable oocytes in female mammals. It is initiated during embryonic development, and it involves the specification of primordial germ cells (PGCs) and progresses through the activation of the meiotic program, reaching a crucial phase in prophase I before pausing at diplotene around the time of birth. The significance of meiosis, particularly the prophase I stage, cannot be overstated, as it plays a pivotal role in ensuring the formation of healthy gametes, a prerequisite for successful reproduction. While research has explored meiosis across various organisms, understanding how environmental factors, including radiation, drugs, endocrine disruptors, reproductive age, or diet, influence this complex developmental process remains incomplete. In this chapter, we describe an ex vivo culture method to investigate meiotic prophase I and beyond and the disruption of oogenesis by external factors. Using this methodology, it is possible to evaluate the effects of individual xenobiotics by administering chemicals at specific points during oogenesis. This culture technique was optimized to study the effects of two selected endocrine disruptors (vinclozolin and MEHP), demonstrating that vinclozolin exposure delayed meiotic differentiation and MEHP exposure reduced follicle size. This approach also opens avenues for future applications, involving the exploration of established or novel pharmaceutical substances and their influence on essential events during prophase I, such as homologous recombination and chromosome segregation. These processes collectively dictate the ultimate fitness of oocytes, with potential implications for factors relevant to the reproductive age and fertility.
Assuntos
Meiose , Ovário , Animais , Feminino , Camundongos , Ovário/citologia , Meiose/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Prófase Meiótica I/efeitos dos fármacos , Disruptores Endócrinos/farmacologia , Oxazóis/farmacologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacosRESUMO
Recently, we reported that, in the naked mole-rat (Heterocephalus glaber) ovary, there is mitotic expansion of the primordial germ cells (PGCs), and the initiation of the meiotic program occurs postnatally. This is opposite to almost all other mammals, including humans and mice, whose reproductive cycle begins very early in development. In both mouse and human, the ovaries become populated with PGCs in utero; these PGCs will later generate the oogonia. After mitotic proliferation, these cells will trigger the meiotic program and initiate meiotic prophase I. Given that all these processes happen in utero, their analysis has been very challenging; so the ability to study them postnatally and to manipulate them with inhibitors or other substances, in the naked mole-rat, opens new possibilities in the field. In this chapter, we present a comprehensive collection of protocols that permit the culture of whole naked mole-rat ovaries, followed by analysis of germ cells, from PGCs to oocytes, in meiotic prophase I, as well the obtention of single-cell suspension or single-nuclei suspension for RNASeq.
Assuntos
Prófase Meiótica I , Ratos-Toupeira , Ovário , Análise de Célula Única , Feminino , Animais , Ovário/citologia , Análise de Célula Única/métodos , Oócitos/citologia , Oócitos/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Meiose , Análise de Sequência de RNA/métodos , Análise da Expressão Gênica de Célula ÚnicaRESUMO
The female reproductive system is essential to women's health, human reproduction and societal well-being. However, the clinical translation of traditional research models is restricted due to the uncertain effects and low efficiency. Emerging evidence shows that microfluidic chips provide valuable platforms for studying the female reproductive system, while no paper has ever comprehensively discussed the topic. Here, a total of 161 studies out of 14,669 records are identified in PubMed, Scopus, Web of Science, ScienceDirect and IEEE Xplore databases. Among these, 61 studies focus on oocytes, which further involves culture, cell surgeries (oocyte separation, rotation, enucleation, and denudation), evaluation and cryopreservation. Forty studies investigate embryo manipulation via microfluidic chips, covering in vitro fertilization, cryopreservation and functional evaluation. Forty-six studies reconstitute both the physiological and pathological statuses of in vivo organs, mostly involved in placenta and fetal membrane research. Fourteen studies perform drug screening and toxicity testing. In this review, we summarize the current application of microfluidic chips in studying the female reproductive system, the advancements in materials and methods, and discuss the future challenges. The present evidence suggests that microfluidic chips-assisted reproductive system reconstruction is promising and more studies are urgently needed.