RESUMO
PURPOSE: To elucidate the effect of X-ray exposure during hysterosalpingography (HSG) on subsequent laboratory outcomes in in vitro fertilization (IVF). METHODS: A total of 1458 oocytes, consisting of 990 oocytes retrieved from 70 women (89 cycles) who underwent HSG prior to IVF and 468 oocytes from 45 women (57 cycles) who underwent IVF without HSG, were evaluated for their retrieval number, maturity, fertilization, and development post fertilization. X-ray exposure during HSG was recorded as reference air kerma (RAK) (mGy). Subjects were stratified according to the amount of RAK (Nil: IVF without HSG, L-RAK: RAK < 16.23, mH-RAK: RAK ≥ 16.23). The number of oocytes retrieved, oocyte maturation, fertilization, and embryo development was compared among 3 groups. Further, multivariate analyses were performed to investigate the effect of X-ray exposure on laboratory outcomes in IVF. RESULTS: There was a statistically significant difference in the fertilization rate among 3 groups (Nil: 71.6%, L-RAK: 80.5%, mH-RAK: 78.3%). The good-quality blastocyst rate in mH-RAK (46.2%) was significantly higher than L-RAK (35.3%) and Nil (32.4%). Multivariate analyses revealed that X-ray exposure was associated with higher fertilization, higher blastocyst development, and higher good-quality blastocyst development rates with adjustment for patient age, BMI, ovarian stimulation types, and fertilization methods. Association between X-ray exposure and the number of oocytes retrieved, and oocyte maturation was not confirmed. CONCLUSIONS: The present study suggests that X-ray exposure of the female reproductive organs during HSG could enhance the potential of oocytes rather than adversely.
Assuntos
Histerossalpingografia/efeitos adversos , Oócitos/efeitos da radiação , Raios X/efeitos adversos , Adulto , Coeficiente de Natalidade , Blastocisto/efeitos da radiação , Desenvolvimento Embrionário/efeitos da radiação , Feminino , Fertilização in vitro/efeitos da radiação , Humanos , Nascido Vivo , Masculino , Recuperação de Oócitos/métodos , Indução da Ovulação/métodos , Gravidez , Taxa de GravidezRESUMO
RESEARCH QUESTION: What is the impact of radiation exposure on oocyte quality and female fertility? DESIGN: Prepubertal mice underwent whole-body irradiation with a single dose (0.02, 0.1, 0.5, 2, 8 Gy) of gamma- or X-rays. Oocytes were quantified in irradiated (nâ¯=â¯36) and sham-treated (nâ¯=â¯8) mice. After a single exposure to 2 Gy, formation of DNA double-strand breaks (nâ¯=â¯10), activation of checkpoint kinase (Chk2) (nâ¯=â¯10) and dynamics of follicular growth (nâ¯=â¯18) were analysed. Fertility assessment was performed in adult irradiated mice and controls from the number of pups per mouse (nâ¯=â¯28) and the fetal abortion rate (nâ¯=â¯24). Ploidy of mature oocytes (nâ¯=â¯20) was analysed after CREST immunostaining, and uterine sections were examined. RESULTS: Radiation exposure induced a massive loss of primordial follicles with LD50 below 50 mGy for both gamma and X-rays. Growing follicles survived doses up to 8 Gy. This difference in radiosensitivity was not due to a different amount of radio-induced DNA damage, and Chk2 was activated in all oocytes. Exposure to a 2 Gy dose abolished the long-term fertility of females due to depletion of the ovarian reserve. Detailed analysis indicates that surviving oocytes were able to complete folliculogenesis and could be fertilized. This transient fertility allowed irradiated females to produce a single litter albeit with a high rate of fetal abortion (23%, Pâ¯=â¯0.0096), related to altered ploidy in the surviving oocytes (25.5%, Pâ¯=â¯0.0035). CONCLUSIONS: The effects of radiation on surviving oocyte quality question natural conception as a first-line approach in cancer survivors. Together, the data emphasize the need for fertility preservation before radiation exposure and call for reassessment of the use of cryopreserved oocytes.
Assuntos
Preservação da Fertilidade/métodos , Oócitos/fisiologia , Oócitos/efeitos da radiação , Ovário/efeitos da radiação , Insuficiência Ovariana Primária/etiologia , Aborto Espontâneo , Aneuploidia , Animais , DNA/efeitos da radiação , Dano ao DNA , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Feminino , Raios gama , Camundongos , Camundongos Endogâmicos C57BL , Folículo Ovariano/efeitos da radiação , Reserva Ovariana/efeitos da radiação , Maturidade Sexual/efeitos da radiação , Irradiação Corporal Total , Raios XRESUMO
Meiotic recombination plays dual roles in the evolution and stable inheritance of genomes: Recombination promotes genetic diversity by reassorting variants, and it establishes temporary connections between pairs of homologous chromosomes that ensure their future segregation. Meiotic recombination is initiated by generation of double-strand DNA breaks (DSBs) by the conserved topoisomerase-like protein Spo11. Despite strong conservation of Spo11 across eukaryotic kingdoms, auxiliary complexes that interact with Spo11 complexes to promote DSB formation are poorly conserved. Here, we identify DSB-3 as a DSB-promoting protein in the nematode Caenorhabditis elegans Mutants lacking DSB-3 are proficient for homolog pairing and synapsis but fail to form crossovers. Lack of crossovers in dsb-3 mutants reflects a requirement for DSB-3 in meiotic DSB formation. DSB-3 concentrates in meiotic nuclei with timing similar to DSB-1 and DSB-2 (predicted homologs of yeast/mammalian Rec114/REC114), and DSB-1, DSB-2, and DSB-3 are interdependent for this localization. Bioinformatics analysis and interactions among the DSB proteins support the identity of DSB-3 as a homolog of MEI4 in conserved DSB-promoting complexes. This identification is reinforced by colocalization of pairwise combinations of DSB-1, DSB-2, and DSB-3 foci in structured illumination microscopy images of spread nuclei. However, unlike yeast Rec114, DSB-1 can interact directly with SPO-11, and in contrast to mouse REC114 and MEI4, DSB-1, DSB-2, and DSB-3 are not concentrated predominantly at meiotic chromosome axes. We speculate that variations in the meiotic program that have coevolved with distinct reproductive strategies in diverse organisms may contribute to and/or enable diversification of essential components of the meiotic machinery.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Quebras de DNA de Cadeia Dupla , Meiose/fisiologia , Animais , Proteínas de Caenorhabditis elegans/genética , Biologia Computacional , Engenharia Genética , Genoma , Oócitos/efeitos da radiaçãoRESUMO
Tethered photoswitches are molecules with two photo-dependent isomeric forms, each with different actions on their biological targets. They include reactive chemical groups capable of covalently binding to their target. Our aim was to develop a ß-subunit-tethered propofol photoswitch (MAP20), as a tool to better study the mechanism of anesthesia through the GABAA α1ß3γ2 receptor. We used short spacers between the tether (methanethiosulfonate), the photosensitive moiety (azobenzene), and the ligand (propofol), to allow a precise tethering adjacent to the putative propofol binding site at the ß+α- interface of the receptor transmembrane helices (TMs). First, we used molecular modeling to identify possible tethering sites in ß3TM3 and α1TM1, and then introduced cysteines in the candidate positions. Two mutant subunits [ß3(M283C) and α1(V227C)] showed photomodulation of GABA responses after incubation with MAP20 and illumination with lights at specific wavelengths. The α1ß3(M283C)γ2 receptor showed the greatest photomodulation, which decreased as GABA concentration increased. The location of the mutations that produced photomodulation confirmed that the propofol binding site is located in the ß+α- interface close to the extracellular side of the transmembrane helices. Tethering the photoswitch to cysteines introduced in the positions homologous to ß3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not entirely reversible, probably reflecting the different nature of each interface. The results are in agreement with a binding site in the ß+α- interface for the anesthetic propofol.
Assuntos
Anestésicos Intravenosos/farmacologia , Membrana Celular/metabolismo , Luz , Oócitos/metabolismo , Propofol/farmacologia , Receptores de GABA-A/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos da radiação , Humanos , Oócitos/efeitos dos fármacos , Oócitos/efeitos da radiação , Conformação Proteica , Domínios Proteicos , Receptores de GABA-A/química , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/efeitos da radiação , Xenopus laevis , Ácido gama-AminobutíricoRESUMO
Hereditary DNA repair defects affect tissues differently, suggesting that in vivo cells respond differently to DNA damage. Knowledge of the DNA damage response, however, is largely based on in vitro and cell culture studies, and it is currently unclear whether DNA repair changes depending on the cell type. Here, we use in vivo imaging of the nucleotide excision repair (NER) endonuclease ERCC-1/XPF-1 in C. elegans to demonstrate tissue-specific NER activity. In oocytes, XPF-1 functions as part of global genome NER (GG-NER) to ensure extremely rapid removal of DNA-helix-distorting lesions throughout the genome. In contrast, in post-mitotic neurons and muscles, XPF-1 participates in NER of transcribed genes only. Strikingly, muscle cells appear more resistant to the effects of DNA damage than neurons. These results suggest a tissue-specific organization of the DNA damage response and may help to better understand pleiotropic and tissue-specific consequences of accumulating DNA damage.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Animais , Caenorhabditis elegans , Feminino , Músculos/metabolismo , Músculos/efeitos da radiação , Neurônios/metabolismo , Neurônios/efeitos da radiação , Oócitos/metabolismo , Oócitos/efeitos da radiação , Especificidade de Órgãos , Raios UltravioletaRESUMO
Germ cells specified during fetal development form the foundation of the mammalian germline. These primordial germ cells (PGCs) undergo rapid proliferation, yet the germline is highly refractory to mutation accumulation compared with somatic cells. Importantly, while the presence of endogenous or exogenous DNA damage has the potential to impact PGCs, there is little known about how these cells respond to stressors. To better understand the DNA damage response (DDR) in these cells, we exposed pregnant mice to ionizing radiation (IR) at specific gestational time points and assessed the DDR in PGCs. Our results show that PGCs prior to sex determination lack a G1 cell cycle checkpoint. Additionally, the response to IR-induced DNA damage differs between female and male PGCs post-sex determination. IR of female PGCs caused uncoupling of germ cell differentiation and meiotic initiation, while male PGCs exhibited repression of piRNA metabolism and transposon derepression. We also used whole-genome single-cell DNA sequencing to reveal that genetic rescue of DNA repair-deficient germ cells (Fancm-/- ) leads to increased mutation incidence and biases. Importantly, our work uncovers novel insights into how PGCs exposed to DNA damage can become developmentally defective, leaving only those genetically fit cells to establish the adult germline.
Assuntos
Dano ao DNA , DNA/efeitos da radiação , Células Germinativas Embrionárias/efeitos da radiação , Células Germinativas/efeitos da radiação , Mutação/genética , Radiação Ionizante , Animais , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/efeitos da radiação , Elementos de DNA Transponíveis/efeitos da radiação , Células Germinativas Embrionárias/citologia , Feminino , Masculino , Meiose/genética , Meiose/efeitos da radiação , Camundongos , Oócitos/citologia , Oócitos/efeitos da radiação , Gravidez , RNA Interferente Pequeno/metabolismo , Fatores SexuaisRESUMO
The exposure of germ cells to radiation introduces mutations in the genomes of offspring, and a previous whole-genome sequencing study indicated that the irradiation of mouse sperm induces insertions/deletions (indels) and multisite mutations (clustered single nucleotide variants and indels). However, the current knowledge on the mutation spectra is limited, and the effects of radiation exposure on germ cells at stages other than the sperm stage remain unknown. Here, we performed whole-genome sequencing experiments to investigate the exposure of spermatogonia and mature oocytes. We compared de novo mutations in a total of 24 F1 mice conceived before and after the irradiation of their parents. The results indicated that radiation exposure, 4 Gy of gamma rays, induced 9.6 indels and 2.5 multisite mutations in spermatogonia and 4.7 indels and 3.1 multisite mutations in mature oocytes in the autosomal regions of each F1 individual. Notably, we found two types of deletions, namely, small deletions (mainly 1~12 nucleotides) in non-repeat sequences, many of which showed microhomology at the breakpoint junction, and single-nucleotide deletions in mononucleotide repeat sequences. The results suggest that these deletions and multisite mutations could be a typical signature of mutations induced by parental irradiation in mammals.
Assuntos
Genoma , Mutação , Oócitos/fisiologia , Espermatogônias/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Raios gama , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Taxa de Mutação , Oócitos/efeitos da radiação , Efeitos da Radiação , Radiação Ionizante , Espermatogônias/efeitos da radiação , Sequenciamento Completo do GenomaRESUMO
Standard cytotoxic cancer treatments, such as radiation, can damage and deplete the supply of oocytes stored within the ovary, which predisposes females to infertility and premature menopause later in life. The mechanisms by which radiation induces oocyte damage have not been completely elucidated. The objective of this study was to determine if γ-irradiation changes mitochondrial characteristics in oocytes, possibly contributing to a reduction in oocyte number and quality. Immature oocytes were collected from postnatal day (PN) 9-11 C57Bl6 mice 3, 6 and 24 hours after 0.1 Gy γ-irradiation to monitor acute mitochondrial changes. Oocytes were classified as small (>20 µm) or growing (40-60 µm). Mitochondrial membrane potential was lost in 20% and 44% of small oocytes (~20 µm) at 3 and 6 hours after γ-irradiation, respectively, consistent with the induction of apoptosis. However, mitochondrial mass, distribution and membrane potential in the surviving small oocytes were similar to the non-irradiated controls at both time points. At 24 hours after γ-irradiation, all mitochondrial parameters analysed within immature oocytes were similar to untreated controls. Mitochondrial parameters within growing oocytes were also similar to untreated controls. When mice were superovulated more than 3 weeks after γ-irradiation, there was a significant reduction in the number of mature oocytes harvested compared to controls (Control 18 ± 1 vs 0.1 Gy 4 ± 1, n = 6/16 mice, p < 0.05). There was a slight reduction in mitochondrial mass in mature oocytes after γ-irradiation, though mitochondrial localization, mtDNA copy number and ATP levels were similar between groups. In summary, this study shows that γ-irradiation of pre-pubertal mice is associated with loss of mitochondrial membrane potential in a significant proportion of small immature oocytes and a reduction in the number of mature oocytes harvested from adult mice. Furthermore, these results suggest that immature oocytes that survive γ-irradiation and develop through to ovulation contain mitochondria with normal characteristics. Whether the oocytes that survive radiation and eventually undergo meiosis can support fertility remains to be determined.
Assuntos
Raios gama/efeitos adversos , Mitocôndrias/efeitos da radiação , Oócitos/efeitos da radiação , Animais , DNA Mitocondrial/genética , Feminino , Fertilidade/efeitos da radiação , Meiose , Potencial da Membrana Mitocondrial/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Oócitos/metabolismo , Ovário/metabolismo , Ovulação/efeitos da radiaçãoRESUMO
Laser based spectroscopic methods can be versatile tools in investigating early stage mammalian embryo structure and biochemical processes in live oocytes and embryos. The limiting factor for using the laser methods in embryological studies is the effect of laser irradiation on the ova. The aim of this work is to explore the optimal parameters of the laser exposure in Raman spectroscopic measurements applicable for studying live early embryos in vitro without impacting their developmental capability. Raman spectra from different areas of mouse oocytes and 2-cells embryos were measured and analyzed. The laser power and exposure time were varied and further embryo development was evaluated to select optimal conditions of the measurements. This work demonstrates safe laser irradiation parameters can be selected, which allow acquisition of Raman spectra suitable for further analysis without affecting the early mouse embryo development in vitro up to morphologically normal blastocyst. The estimation of living embryo state is demonstrated via analysis and comparison of the spectra from fertilized embryo with the spectra from unfertilized oocytes or embryos subjected to UV laser irradiation. These results demonstrate the possibility of investigating preimplantation mammalian embryo development and estimating its state/quality. It will have potential in developing prognosis of mammalian embryos in assisted reproductive technologies.
Assuntos
Blastocisto/efeitos da radiação , Embrião de Mamíferos/efeitos da radiação , Oócitos/efeitos da radiação , Análise Espectral Raman/métodos , Animais , Desenvolvimento Embrionário/efeitos da radiação , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Raios UltravioletaRESUMO
Purpose: To review the history of radiation effects in germ cells and to attempt to discern future directions. Materials and methods: Review of published papers. Results: Recent mouse studies using microarray-based comparative genomic hybridization techniques revealed that germline mutations would occur much less frequently than would be expected from the results of past studies which used specific locus tests. The mean induction rate of deletion mutations was about 1%/genome per Gy. In other words, one out of 100 offspring following a parental (possibly only paternal) exposure to 1 Gy may be expected to have one deletion. This means that at low doses, the genetic risk of radiation is so small and difficult to detect, especially when we learn that our genomes are not perfect and carry as many as on average 80 inactivated genes under heterozygous conditions. Nevertheless, any radiation-induced de novo mutations can be deletions which may involve haploid-sensitive genes, and hence can be a threat to health. Conclusion: Although powerful methods such as whole-genome-sequencing techniques have become available to detect radiation effects in human germ cells, the mutation induction rate in the genome now appears to be much lower than was previously thought. Consequently, detecting radiation effects in human germ cells still remains a difficult task.
Assuntos
Hibridização Genômica Comparativa , Lesões por Radiação/genética , Lesões por Radiação/história , Animais , Aberrações Cromossômicas/efeitos da radiação , Reparo do DNA , Deleção de Genes , Genoma Humano , Células Germinativas , Mutação em Linhagem Germinativa , Haploidia , Heterozigoto , História do Século XX , História do Século XXI , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos/efeitos da radiação , Fenótipo , Liberação Nociva de Radioativos , RiscoRESUMO
Xenopus laevis oocytes are a widely used model system because of their capacity to translate exogenous mRNA, but their high intrinsic background fluorescence is a disadvantage for fluorescence recordings. Here, we developed two distinct methods for improving fluorescence recordings from oocytes. One was a pharmacological method in which a small-molecule salt-inducible kinase inhibitor was co-injected with the mRNA of interest to stimulate melanin production. We interrogated the oocytes using cut-open voltage clamp with simultaneous fluorescence recording and found that by increasing the amount of light-absorbing melanin in these oocytes, we decreased their intrinsic background fluorescence. The treated oocytes produced fluorescence signals that were approximately four times larger. The second method consisted of direct injection of synthetic melanin. This method also significantly improved (doubled) fluorescence signals and allowed any oocyte to be used for fluorescence recording. These two methods provide significant improvements of the signal quality for fluorescent oocyte recordings and allow all healthy oocytes to be used for high-sensitivity recordings.
Assuntos
Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Animais , Feminino , Fluorescência , Melaninas/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/efeitos da radiação , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Raios Ultravioleta , XenopusRESUMO
Here, we aimed to identify biomarkers for mice oocyte maturation in metaphase II in vivo and in situ using Raman spectroscopy. Principal component analysis of 324 Raman data points of oocytes at Phase I, II, III, and IV showed that the phosphoric acid concentration uniformly increased in oocytes with higher developmental competence than in oocytes at other maturation stages, and proteins were more phosphorylated. The maturation phases were successfully predicted by linear discriminant analysis with high accuracy (90.7%) using phosphoric molecular information mentioned above. Furthermore, detections of higher concentration of unsaturated fatty acids in overmatured oocytes indicated that a decline in metabolic activity due to overmaturation induced a surplus of these lipid components. Upon assessing invasiveness by laser irradiation, about 50% irradiated oocytes progressed to morula and blastocyst stages in good conditions. Thus, Raman spectroscopy holds promise in evaluating oocyte maturation and quality based on molecular information in infertility treatment.
Assuntos
Oócitos/crescimento & desenvolvimento , Ácidos Fosfóricos/análise , Animais , Biomarcadores/análise , Blastocisto/metabolismo , Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Análise Discriminante , Feminino , Cavalos , Humanos , Raios Infravermelhos , Lipídeos/análise , Masculino , Fator Promotor de Maturação/metabolismo , Camundongos Endogâmicos ICR , Mórula/metabolismo , Oócitos/química , Oócitos/classificação , Oócitos/efeitos da radiação , Fosforilação , Gravidez , Análise de Componente Principal , Análise Espectral RamanRESUMO
Purpose: To reflect on the contributions of R. H. Mole to radiobiology. Robin Mole was a very active radiobiologist when the IJRB was first established and had two papers in the first few issues of the journal in 1959. Conclusions: R. H. Mole made significant contributions to radiobiology and is particularly associated with studies of the effects of low dose exposures on the reproductive health of mammals. Many of his ideas are still debated and remain controversial.
Assuntos
Radiobiologia/história , Saúde Reprodutiva , Animais , Carcinogênese , Feminino , História do Século XX , Humanos , Mamíferos , Neoplasias/etiologia , Neoplasias Induzidas por Radiação , Oócitos/efeitos da radiação , Reprodução/efeitos da radiaçãoRESUMO
Fluorescence photoactivation provides a strategy for monitoring protein kinetics within living cells. In particular, fluorescence photoactivation of a subpopulation of microtubule subunits within the spindle using photoactivatable fluorescent tubulin constructs has proven useful for assessing a variety of features of spindle microtubule dynamics, including poleward microtubule movement, microtubule depolymerization, and microtubule turnover, in various cellular settings. The current chapter describes a method for monitoring microtubule dynamics within the mouse egg spindle by photoactivation of photoactivatable-GFP-tubulin, followed by time-lapse confocal imaging.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Microtúbulos/metabolismo , Oócitos/fisiologia , Tubulina (Proteína)/metabolismo , Animais , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/efeitos da radiação , Camundongos , Microscopia de Fluorescência , Oócitos/citologia , Oócitos/efeitos da radiação , Processos Fotoquímicos , Fármacos Fotossensibilizantes , Fuso Acromático/metabolismo , Imagem com Lapso de Tempo , Tubulina (Proteína)/genéticaRESUMO
Development of fluorescence distribution assays like FRAP (fluorescence recovery after photobleaching) or photoactivation has had a great impact in studying intracellular protein dynamics. In particular, the cytoskeleton field largely benefited from these techniques, with lots of new information provided about the dynamics and organization of actin networks whithin cells.In mouse oocyte, actin photoactivation has been very useful to determine the dynamics of different actin structures involved in meiotic divisions, including a cytoplasmic meshwork and a subcortical actin layer.Here, we describe a method, actin photoactivation, to determine the dynamics of the actin cytoplasmic meshwork and the subcortical actin layer during the first meiotic division in the mouse oocyte, that could be adapted to other actin structures or other stages of meiotic divisions.
Assuntos
Actinas/metabolismo , Recuperação de Fluorescência Após Fotodegradação/métodos , Proteínas de Fluorescência Verde/metabolismo , Meiose , Oócitos/metabolismo , Actinas/efeitos da radiação , Animais , Citoplasma/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/efeitos da radiação , Camundongos , Oócitos/citologia , Oócitos/efeitos da radiaçãoRESUMO
Laser ablation is a powerful tool to study forces within biological systems. This technique has been extensively used to study mitotic spindle formation and chromosome segregation. This chapter describes laser ablation of microtubule-chromosome attachments coupled to fluorescence live microscopy and quantitative analysis of individual chromosome movement after microtubule severing. This method allows to gain insight into the organization and dynamics of the meiotic spindle and chromosomes in metaphase I mouse oocytes.
Assuntos
Segregação de Cromossomos , Terapia a Laser/métodos , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Oócitos/fisiologia , Animais , Feminino , Meiose , Camundongos , Oócitos/citologia , Oócitos/efeitos da radiação , Fuso Acromático/metabolismoRESUMO
The negative effects of ambient ultraviolet (UVA) on the water environment have been recently highlighted; UVA can create deleterious effects by stimulating stress on pelagic organisms. Little is known about UVA effects on oocyte characteristics of female fish. In the present study we explored the effects of exposure to ecologically relevant levels of simulated UVA radiation on ovaries of two major strains WT (HdrR) and P53 (-/-) of medaka (Oryzias latipes) mature female. Fish were assigned to control and three UVA-exposed groups as (15â¯min, 30â¯min, and 60â¯min/day) for three days and sample selection was 24â¯h and 14â¯days after exposure. Histological alterations and oocyte atresia percentage were analyzed in the UVA-exposed fish compared to control. Alteration comprised hyperthrophied follicular cells with increased thickness, breakdown of egg chorion (zona radiata), damage of cortical alveoli, and distorted nucleus and cytoplasm. The atresia percentages significantly increased with higher UVA exposure dose and time for both the wild type and the p53 deficient fish. The wild type displayed significantly higher oocyte atresia percentage than the p53 mutant. These results suggested that UVA exposure provoked histological alterations in both p53 and WT medaka oocytes leading to follicular atresia, which reduce female reproductive ability and larval production. UVA oocyte response showed p53 dependent and independent histological alteration, however, the p53 mutant was less sensitive to UVA than the wild type in medaka fish.
Assuntos
Raios Ultravioleta , Vitelogênese/efeitos da radiação , Animais , Feminino , Proteínas de Peixes/deficiência , Proteínas de Peixes/genética , Oócitos/metabolismo , Oócitos/efeitos da radiação , Oryzias/genética , Oryzias/crescimento & desenvolvimento , Ovário/patologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Ciliary opsins were classically thought to function only in vertebrates for vision, but they have also been identified recently in invertebrates for non-visual photoreception. Larvae of the annelid Platynereis dumerilii are used as a zooplankton model, and this zooplankton species possesses a "vertebrate-type" ciliary opsin (named c-opsin) in the brain. Platynereis c-opsin is suggested to relay light signals for melatonin production and circadian behaviors. Thus, the spectral and biochemical characteristics of this c-opsin would be directly related to non-visual photoreception in this zooplankton model. Here we demonstrate that the c-opsin can sense UV to activate intracellular signaling cascades and that it can directly bind exogenous all-trans-retinal. These results suggest that this c-opsin regulates circadian signaling in a UV-dependent manner and that it does not require a supply of 11-cis-retinal for photoreception. Avoidance of damaging UV irradiation is a major cause of large-scale daily zooplankton movement, and the observed capability of the c-opsin to transmit UV signals and bind all-trans-retinal is ideally suited for sensing UV radiation in the brain, which presumably lacks enzymes producing 11-cis-retinal. Mutagenesis analyses indicated that a unique amino acid residue (Lys-94) is responsible for c-opsin-mediated UV sensing in the Platynereis brain. We therefore propose that acquisition of the lysine residue in the c-opsin would be a critical event in the evolution of Platynereis to enable detection of ambient UV light. In summary, our findings indicate that the c-opsin possesses spectral and biochemical properties suitable for UV sensing by the zooplankton model.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Opsinas/metabolismo , Células Fotorreceptoras de Invertebrados/efeitos da radiação , Poliquetos/fisiologia , Sistemas do Segundo Mensageiro/efeitos da radiação , Zooplâncton/fisiologia , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Cílios/metabolismo , Cílios/efeitos da radiação , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Lisina/química , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Oócitos/metabolismo , Oócitos/efeitos da radiação , Opsinas/química , Opsinas/genética , Técnicas de Patch-Clamp , Células Fotorreceptoras de Invertebrados/metabolismo , Filogenia , Poliquetos/efeitos da radiação , Estabilidade Proteica/efeitos da radiação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Retinaldeído/química , Retinaldeído/metabolismo , Estereoisomerismo , Raios Ultravioleta , Xenopus , Zooplâncton/efeitos da radiaçãoRESUMO
The purpose of this study was to evaluate the effects of 3rd-generation (3G) cellular phone radiofrequency-electromagnetic wave (RF-EMW) exposure on fertilization and embryogenesis in mice. Oocytes and spermatozoa were exposed to 3G cellular phone RF-EMWs, 1.95 GHz wideband code division multiple access, at a specific absorption rate of 2 mW/g for 60 min, or to sham exposure. After RF-EMW exposure, in vitro fertilization and intracytoplasmic sperm injection were performed. Rates of fertilization, embryogenesis (8-cell embryo, blastocyst), and chromosome aberration were compared between the combined spermatozoa and oocyte groups: both exposed, both non-exposed, one exposed, and the other non-exposed. Rates of fertilization, embryogenesis, and blastocyst formation did not change significantly across the four groups. Considering that the degree of exposure in the present study was ≥100 times greater than daily exposure of human spermatozoa and even greater than daily exposure of oocytes, the present results indicate safety of RF-EMW exposure in humans. Bioelectromagnetics. 38:466-473, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Telefone Celular , Desenvolvimento Embrionário/efeitos da radiação , Ondas de Rádio/efeitos adversos , Injeções de Esperma Intracitoplásmicas/efeitos da radiação , Animais , Aberrações Cromossômicas/efeitos da radiação , Feminino , Masculino , Camundongos , Oócitos/fisiologia , Oócitos/efeitos da radiação , Espermatozoides/fisiologia , Espermatozoides/efeitos da radiaçãoRESUMO
Ovarian function is directly correlated with survival of the primordial follicle reserve. Women diagnosed with cancer have a primary imperative of treating the cancer, but since the resting oocytes are hypersensitive to the DNA-damaging modalities of certain chemo- and radiotherapeutic regimens, such patients face the collateral outcome of premature loss of fertility and ovarian endocrine function. Current options for fertility preservation primarily include the collection and cryopreservation of oocytes or in vitro-fertilized oocytes, but this necessitates a delay in cancer treatment and additional assisted reproductive technology procedures. Here, we evaluated the potential of pharmacological preservation of ovarian function by inhibiting a key element of the oocyte DNA damage checkpoint response, checkpoint kinase 2 (CHK2; CHEK2). Whereas nonlethal doses of ionizing radiation (IR) eradicate immature oocytes in wild-type mice, irradiated Chk2-/- mice retain their oocytes and, thus, fertility. Using an ovarian culture system, we show that transient administration of the CHK2 inhibitor 2-(4-(4-chlorophenoxy)phenyl)-1H-benzimidazole-5-carboxamide-hydrate ("CHK2iII") blocked activation of the CHK2 targets TRP53 and TRP63 in response to sterilizing doses of IR, and preserved oocyte viability. After transfer into sterilized host females, these ovaries proved functional and readily yielded normal offspring. These results provide experimental evidence that chemical inhibition of CHK2 is a potentially effective treatment for preserving the fertility and ovarian endocrine function of women exposed to DNA-damaging cancer therapies such as IR.