RESUMO
During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n=27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P<0.05). The proliferation index and numerical density of proliferating cells in the stroma significantly decreased (P<0.05), whereas the numerical density of cells in the stroma did not change (P>0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P<0.05) and the expression of many genes was positively correlated with other genes and with gestational age. Thus, the rate of cortical stromal expansion peaked in early gestation due to cell proliferation, whilst late in development expression of extracellular matrix genes increased.
Assuntos
Proliferação de Células/fisiologia , Expressão Gênica , Folículo Ovariano/crescimento & desenvolvimento , Ovário/crescimento & desenvolvimento , Animais , Bovinos , Colágeno Tipo I/metabolismo , Feminino , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ovário/citologia , Ovário/metabolismoRESUMO
In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand breaks (DSBs), a process that precedes homologous synapsis. Here we use an antibody specific for a phosphorylated histone (gamma-H2AX, which marks the sites of DSBs) to investigate the timing, distribution and Spo11-dependence of meiotic DSBs in the mouse. We show that, as in yeast, recombination in the mouse is initiated by Spo11-dependent DSBs that form during leptotene. Loss of gamma-H2AX staining (which in irradiated somatic cells is temporally linked with DSB repair) is temporally and spatially correlated with synapsis, even when this synapsis is 'non-homologous'.
Assuntos
DNA/genética , Integrases , Meiose/genética , Recombinação Genética , Animais , Anticorpos , Proteínas de Ciclo Celular , DNA/metabolismo , DNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a DNA , Endodesoxirribonucleases , Esterases/genética , Esterases/metabolismo , Feminino , Histonas/imunologia , Histonas/metabolismo , Masculino , Meiose/fisiologia , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Proteínas/genética , Proteínas/metabolismo , Recombinases , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Nuclear contents or cytoplasm from Xenopus oocytes labeled with (35-S)methionine or (3-H)proline (donor oocytes) were reinjected into unlabeled oocytes (recipient oocytes). The radioactivity injected as nuclear contents was found to enter and accumulate in the recipient oocyte nucleus. In contrast, the radioactivity injected as cytoplasm was found to enter but not to accumulate in the recipient oocyte nucleus. Sodium dodecyl sulfate (SDS) gel electrophoresis of the nucleus and cytoplasm of donor oocytes revealed the existence of three classes of labeled proteins in these oocytes: those proteins found predominantly in the nucleus (N proteins), those found predominantly in the cytoplasm (C proteins), and those found in both the nucleus and cytoplasm at similar concentrations (B proteins). SDS gel electrophoresis of the nucleus and cytoplasm of recipient oocytes showed that N proteins entered and accumulated in the nucleus but that B proteins partitioned about equally between the nucleus and cytoplasm. A similar analysis of oocytes injected with labeled cytoplasm showed that C proteins did not enter the nucleus but again B proteins partitioned about equally between the nucleus and cytoplasm.
Assuntos
Núcleo Celular/metabolismo , Óvulo/metabolismo , Proteínas/metabolismo , Animais , Autorradiografia , Núcleo Celular/análise , Citoplasma/análise , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Metionina/metabolismo , Microinjeções , Micromanipulação , Prolina/metabolismo , Proteínas/análise , Radioisótopos de Enxofre , Trítio , XenopusRESUMO
A technique is presented which enables one to measure the extent to which a protein enters and accumulates in the nucleus of the frog oocyte. In this method, the protein, labeled with 125-I, is microinjected into the oocyte. After incubation, the oocyte is manually enucleated and the radioactivity in the nucleus and cytoplasm is determined. Using this technique, proteins lighter than 20,000 daltons were found to enter the nucleus and completely equilibrate between the nucleus and cytoplasm within 24 h. The entry of proteins heavier than 69,000 daltons was severely hindered. Histones and histone fractions entered as quickly as other small proteins, but, in contrast to these proteins, they accumulated in the nucleus to different extents, depending on the total amount of histone injected into the oocyte and the identity of the histone. Evidence is presented that histone fractions compete with each other for accumulation in the nucleus.
Assuntos
Núcleo Celular/metabolismo , Histonas/metabolismo , Óvulo/metabolismo , Animais , Autorradiografia , Bovinos , Galinhas , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Eritrócitos , Feminino , Radioisótopos do Iodo , Microinjeções , Micromanipulação , Peso Molecular , Muramidase/metabolismo , Mioglobina/metabolismo , Ovalbumina/metabolismo , Pâncreas , Soroalbumina Bovina/metabolismo , Timo , Inibidores da Tripsina/metabolismo , Xenopus , gama-Globulinas/metabolismoRESUMO
The histone 2A proteins of the sea urchin Strongylocentrotus purpuratus are compared with those of the mouse. While the major H2As in these two organisms do not comigrate on two-dimensional gels, the sea urchin contains a protein that comigrates with the minor histone 2A variant H2A.Z from mammals. H2A.Z is of particular interest because its sequence homology with other H2As is quite low, and it is not phosphorylated as are other H2As. A comparison of the tryptic peptide patterns of several H2As from sea urchin blastulae and mouse L1210 cells show that, while the patterns of the H2A.Zs differ greatly from the patterns of the other H2As, the patterns of the mouse and sea urchin H2A.Zs are very similar. Since the H2A.Zs have only one or two peptides in common with the other H2As, the conservation of their sequence indicates that H2A.Zs have evolved under somewhat different selective pressures from other H2As. Unlike all the other sea urchin H2As whose syntheses either turn on or off during early development, H2A.Z seems to be synthesized continuously throughout this period.U
Assuntos
Histonas/análise , Camundongos/metabolismo , Ouriços-do-Mar/análise , Animais , Blastocisto/metabolismo , DNA/biossíntese , Eletroforese em Gel de Poliacrilamida , Gástrula/metabolismo , Histonas/biossíntese , Mórula/metabolismo , Peptídeos/análise , Ouriços-do-Mar/embriologiaRESUMO
The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named gamma-H2AX. An antibody prepared to the unique region of human gamma-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that gamma-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, gamma-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, gamma-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that gamma-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Dano ao DNA/genética , Animais , Western Blotting , Linhagem Celular , Cromatina/imunologia , Cromatina/efeitos da radiação , Cromossomos/genética , Cromossomos/metabolismo , Cromossomos/efeitos da radiação , DNA/genética , DNA/metabolismo , DNA/efeitos da radiação , Dano ao DNA/efeitos da radiação , Fibroblastos , Histonas/química , Histonas/genética , Histonas/imunologia , Histonas/metabolismo , Humanos , Interfase/genética , Interfase/efeitos da radiação , Lasers , Microscopia Confocal , Mitose/genética , Mitose/efeitos da radiação , Cervo Muntjac , Fosforilação/efeitos da radiação , Fosfosserina/metabolismo , Radiação Ionizante , Fatores de TempoRESUMO
Cis and trans dichlorodiaminoplatinum (II) compounds bind to DNA and form DNA cross-links, which are usually considered to be irreversible. Thiourea can reverse these cross-links without any apparent breakdown of the DNA. In addition, cis- and trans-Pt (II) treatment of lambda decreases its transfectivity. After suitable incubation with thiourea, full transfectivity of Pt(II)-treated lambda DNA can be restored.
Assuntos
DNA/metabolismo , Compostos Organoplatínicos/antagonistas & inibidores , Tioureia/farmacologia , Animais , Linhagem Celular , Colífagos , DNA de Neoplasias/metabolismo , DNA Viral/metabolismo , Leucemia L1210 , Relação Estrutura-AtividadeRESUMO
Histone octamers from calf thymus were separated into (H3:H4)2 tetramers and H2A:H2B dimers by chromatography through Sephadex G100. The tetramers and dimers were analyzed for variants, ubiquitin adducts, and proteolyzed forms. The minor histone variants H2A.X and H2A.Z were found to be associated with histone H2B as H2A.X:H2B and H2A.Z:H2B dimers, respectively. Ubiquitin adducts of the H2A's and H2B were also present in H2A:H2B dimers.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Animais , Bovinos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Histonas/isolamento & purificação , UbiquitinasRESUMO
Genetic disorders affecting cellular responses to DNA damage are characterized by high rates of translocations involving antigen receptor loci and increased susceptibility to lymphoid malignancies. We report that the Nijmegen breakage syndrome protein (NBS1) and histone gamma-H2AX, which associate with irradiation-induced DNA double-strand breaks (DSBs), are also found at sites of VDJ (variable, diversity, joining) recombination-induced DSBs. In developing thymocytes, NBS1 and gamma-H2AX form nuclear foci that colocalize with the T cell receptor alpha locus in response to recombination activating gene (RAG) protein-mediated VDJ cleavage. Our results suggest that surveillance of T cell receptor recombination intermediates by NBS1 and gamma-H2AX may be important for preventing oncogenic translocations.
Assuntos
Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Recombinação Genética , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Dados de Sequência Molecular , FosforilaçãoRESUMO
This study provides evidence for the importance of p21(CDKN1A) for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21(CDKN1A) and p53 enhance camptothecin-induced histone H2AX phosphorylation (gammaH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, gammaH2AX reverses after camptothecin removal. By contrast, gammaH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53-/-) or p21(CDKN1A) (p21-/-) as the cells reach the late-S and G2 phases. Since p21-/- cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced gammaH2AX formation in p21-/- cells, we conclude that enhanced gammaH2AX formation in p21-/- cells is not due to re-replication. The cell cycle checkpoint abrogator and Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) also increases camptothecin-induced gammaH2AX formation and inhibits camptothecin-induced p21(CDKN1A) upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that gammaH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21(CDKN1A) prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Reparo do DNA/fisiologia , Replicação do DNA , DNA Topoisomerases Tipo I/farmacologia , DNA/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estaurosporina/análogos & derivados , Afidicolina/farmacologia , Apoptose/efeitos dos fármacos , Quebra Cromossômica , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , DNA/genética , Dano ao DNA/efeitos dos fármacos , Células HCT116 , Histonas/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Fase S/efeitos dos fármacos , Estaurosporina/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
BACKGROUND: The response of eukaryotic cells to double-strand breaks in genomic DNA includes the sequestration of many factors into nuclear foci. Recently it has been reported that a member of the histone H2A family, H2AX, becomes extensively phosphorylated within 1-3 minutes of DNA damage and forms foci at break sites. RESULTS: In this work, we examine the role of H2AX phosphorylation in focus formation by several repair-related complexes, and investigate what factors may be involved in initiating this response. Using two different methods to create DNA double-strand breaks in human cells, we found that the repair factors Rad50 and Rad51 each colocalized with phosphorylated H2AX (gamma-H2AX) foci after DNA damage. The product of the tumor suppressor gene BRCA1 also colocalized with gamma-H2AX and was recruited to these sites before Rad50 or Rad51. Exposure of cells to the fungal inhibitor wortmannin eliminated focus formation by all repair factors examined, suggesting a role for the phosphoinositide (PI)-3 family of protein kinases in mediating this response. Wortmannin treatment was effective only when it was added early enough to prevent gamma-H2AX formation, indicating that gamma-H2AX is necessary for the recruitment of other factors to the sites of DNA damage. DNA repair-deficient cells exhibit a substantially reduced ability to increase the phosphorylation of H2AX in response to ionizing radiation, consistent with a role for gamma-H2AX in DNA repair. CONCLUSIONS: The pattern of gamma-H2AX foci that is established within a few minutes of DNA damage accounts for the patterns of Rad50, Rad51, and Brca1 foci seen much later during recovery from damage. The evidence presented strongly supports a role for the gamma-H2AX and the PI-3 protein kinase family in focus formation at sites of double-strand breaks and suggests the possibility of a change in chromatin structure accompanying double-strand break repair.
Assuntos
Dano ao DNA , Enzimas Reparadoras do DNA , Reparo do DNA , Histonas/metabolismo , Hidrolases Anidrido Ácido , Androstadienos/farmacologia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bromodesoxiuridina/farmacologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Raios gama , Humanos , Lasers , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Rad51 Recombinase , Células Tumorais Cultivadas , Raios Ultravioleta , WortmaninaRESUMO
It has been documented widely that when the generation times of eucaryotic cells are lengthened by slowing the rate of protein synthesis, the duration of the chromosome cycle (S, G2, and M phases) remains relatively invariant. Paradoxically, when the growth of exponentially growing cultures of CHO cells is partially inhibited with inhibitors of protein synthesis, the immediate effect is a proportionate reduction in the rate of total protein, histone protein, and DNA synthesis. However, on further investigation it was found that over the next 2 h the rates of histone protein and DNA synthesis recover, in some cases completely to the uninhibited rate, while the synthesis rates of other proteins do not recover. We called this process chromosome cycle compensation. The amount of compensation seen in CHO cell cultures can account quantitatively for the relative invariance in the length of the chromosome cycle (S, G2, and M phases) reported for these cells. The mechanism for this compensation involves a specific increase in the levels of histone mRNAs. An invariant chromosome cycle coupled with a lengthening growth cycle must result in a disproportionate lengthening of the G1 phase. Thus, these results suggest that chromosome cycle invariance may be due more to specific cellular compensation mechanisms rather than to the more usual interpretation involving a rate-limiting step for cell cycle progression in the G1 phase.
Assuntos
Ciclo Celular , Cromossomos/fisiologia , Biossíntese de Proteínas , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Cricetulus , Cicloeximida/farmacologia , Replicação do DNA/efeitos dos fármacos , Feminino , Histonas/biossíntese , Histonas/genética , Hidroxiureia/farmacologia , Cinética , Ovário , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
Factors in the extracellular environment, specifically hypertonic or acidic growth media, are shown to alter the modification of histones in several cell lines. For histone 2A, changes in modification were visible in the mass pattern and were found to be primarily changes in phosphorylation. The increased modification of the core histones was quickly reversed when cells were returned to normal medium.
Assuntos
Histonas/metabolismo , Leucemia L1210/metabolismo , Acetilação , Animais , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Histonas/isolamento & purificação , Concentração de Íons de Hidrogênio , Camundongos , Peso Molecular , Concentração Osmolar , FosforilaçãoRESUMO
During the replication of chromatin, the syntheses of the histone protein and DNA components are closely coordinated but not totally linked. The interrelationships of total protein synthesis, histone protein synthesis, DNA synthesis, and mRNA levels have been investigated in Chinese hamster ovary cells subjected to several different types of inhibitors in several different temporal combinations. The results from these studies and results reported elsewhere can be brought together into a consistent framework which combines the idea of autoregulation of histone biosynthesis as originally proposed by W. B. Butler and G. C. Mueller (Biochim. Biophys. Acta 294:481-496, 1973] with the presence of basal histone synthesis and the effects of protein synthesis on DNA synthesis. The proposed framework obviates the difficulties of Butler and Mueller's model and may have wider application in understanding the control of cell growth.
Assuntos
Divisão Celular , Cromatina/biossíntese , Replicação do DNA , Histonas/biossíntese , Biossíntese de Proteínas , Animais , Linhagem Celular , Cricetinae , Cricetulus , Replicação do DNA/efeitos dos fármacos , Depressão Química , Feminino , Fibroblastos/metabolismo , Ovário , Biossíntese de Proteínas/efeitos dos fármacos , Fatores de TempoRESUMO
Histone H2A.X is a replication-independent histone H2A isoprotein species that is encoded by a transcript alternatively processed at the 3' end to yield two mRNAs: a 0.6-kb mRNA ending with the stem-loop structure characteristic of the mRNAs for replication-linked histone species, and a second, polyadenylated 1.6-kb mRNA ending about 1 kb further downstream (C. Mannironi, W. M. Bonner, and C. L. Hatch, Nucleic Acids Res. 17:9113-9126, 1989). Of the two, the 0.6-kb H2A.X stem-loop mRNA predominates in many cell lines, indicating that the presence of two types of mRNA may not completely account for the replication independence of H2A.X protein synthesis. The ambiguity is resolved by the finding that the level of the 0.6-kb H2A.X mRNA is only weakly downregulated during the inhibition of DNA replication and only weakly upregulated during the inhibition of protein synthesis, while the levels of other replication-linked mRNAs are strongly down- or upregulated under these two conditions. Analysis of the nuclear transcription rates of specific H2A genes showed that while the rates of transcription of replication-linked H2A genes decreased substantially during the inhibition of DNA synthesis and increased substantially during the inhibition of protein synthesis, the rate of H2A.X gene transcription decreased slightly under both conditions. These differences in transcriptional regulation between the H2A.X gene and other replication-linked histone genes are sufficient to account for the differences in regulation of their respective stem-loop mRNAs.
Assuntos
Replicação do DNA , Regulação da Expressão Gênica , Histonas/genética , Transcrição Gênica , Sequência de Bases , Diferenciação Celular/genética , Linhagem Celular , Citoplasma/metabolismo , DNA , Ligação Genética , Células HeLa , Histonas/metabolismo , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Radiation-induced bystander effect has been well documented. However, the mechanisms are poorly understood. How we incorporate this effect into the classical models of risk assessment remains an open question. Here, the induction of bystander effect was studied by assessing DNA double-strand break (DSB) formation in situ with the rapid and sensitive gamma-H2AX focus formation assay. Utilising the Columbia University single-cell microbeam system to deliver 2 or 20 individual alpha particles to selected cell nuclei in a precisely known proportion of cells in a population, the induced DNA DSB incidences were quantified 30 min and 18 h post-IR. The increase in DNA DSB incidence in bystander cells lacked of a linear dose response indicating that neither the dose of irradiation nor proportion of irradiated cells in a population, is a critical parameter. This study confirms a binary all-or-nothing model of triggering the bystander response. The delay and persistence of the bystander response suggests a different mechanism of DSB induction in bystander cells than in directly irradiated cells.
Assuntos
Efeito Espectador/fisiologia , Efeito Espectador/efeitos da radiação , Dano ao DNA , DNA/genética , DNA/efeitos da radiação , Fibroblastos/fisiologia , Fibroblastos/efeitos da radiação , Partículas alfa , Linhagem Celular , Relação Dose-Resposta à Radiação , Humanos , Doses de Radiação , Tolerância a Radiação/fisiologia , Tolerância a Radiação/efeitos da radiaçãoRESUMO
Human H2AZ gene promoter fragments that included sequences upstream from the core promoter resulted in decreased activity of reporter constructs transfected into several human cell lines, but increased activity in the undifferentiated human embryonal carcinoma cell line Tera-2. Differentiation of Tera-2 cells in media containing retinoic acid restored the ability of the upstream region to downregulate H2AZ gene promoter activity. Levels of endogenous H2AZ mRNA were also found to be 2.5-fold higher in undifferentiated Tera-2 cells than in differentiated Tera-2 cells. A 128 bp region located 234 to 361 bp upstream from the transcription start site of the H2AZ gene was found to be responsible for the modulation of reporter activity. The upstream region also functioned similarly when removed from the H2AZ gene promoter and inserted upstream of the SV40 promoter in reporter constructs. Gel mobility shift studies of fragments of this region revealed two sequence elements, CTCCTCC and CACGTG, that bound nuclear factors in vitro.
Assuntos
Histonas/genética , Regiões Promotoras Genéticas , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , DNA/genética , DNA/metabolismo , Genes Reporter , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Correction for 'X-Ray fluorescence imaging and other analyses identify selenium and GPX1 as important in female reproductive function' by M. J. Ceko et al., Metallomics, 2014, DOI: .
RESUMO
Studies of selenium (Se) status indicate that Se is necessary for fertility but how precisely is not known. We aimed to show that Se was important in bovine female reproductive function. The elemental distribution in the bovine ovary (n = 45 sections) was identified by X-ray fluorescence (XRF) imaging. Se was consistently localized to the granulosa cell layer of large (>10 mm) healthy follicles. Inductively Coupled Plasma - Mass Spectrometry revealed tenfold higher Se in the bovine follicle wall compared to corpora lutea. Gene expression analysis of selenoprotein genes GPX1, GPX3, VIMP and SELM in bovine granulosa cells revealed that only GPX1 was significantly up-regulated in large healthy follicles compared to the small healthy or atretic follicles (P < 0.05). Western immunoblotting identified GPX1 protein in bovine granulosa cells of large healthy follicles, but not of small healthy follicles. To assess if GPX1 was important in human follicles, cumulus cells from women undergoing IVF/ICSI with single embryo transfer were collected. Oocytes and embryos were cultured and transferred independently in 30 patients undergoing elective single embryo transfer. Gene expression of GPX1 was significantly higher in human cumulus cells from cumulus-oocyte complexes yielding a pregnancy (P < 0.05). We present the first XRF imaging of mammalian ovaries showing that Se is consistently localized to the granulosa cells of large healthy follicles. We conclude that Se and selenoproteins are elevated in large healthy follicles and may play a critical role as an antioxidant during late follicular development.
Assuntos
Células do Cúmulo/metabolismo , Glutationa Peroxidase/metabolismo , Folículo Ovariano/metabolismo , Selênio/metabolismo , Animais , Bovinos , Células Cultivadas , Células do Cúmulo/química , Feminino , Perfilação da Expressão Gênica , Glutationa Peroxidase/análise , Glutationa Peroxidase/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Folículo Ovariano/química , Reação em Cadeia da Polimerase , Espectrometria por Raios X , Glutationa Peroxidase GPX1RESUMO
When nuclei were treated with the chemotherapeutic agent, cis-Pt(II), they were crosslinked to the extent that their nuclear morphology as assayed by light microscopy was retained even in the presence of SDS. Protein analysis showed that the histones were completely absent from these nuclear structures, while the non-histone proteins, with one possible exception, were completely retained. When the nuclear structures in SDS were treated with thiourea to reverse the crosslinks, the non-histone proteins were liberated and the nuclear structures disappeared. When treated with Proteinase K in SDS, the nuclear structures also disappeared, indicating that protein components were necessary to maintain the structures.