Chromomycin A3 staining as a useful tool for evaluation of male fertility.
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PURPOSE: Our purpose was to investigate the association between percentage chromomycin A3 (CMA3) positivity of spermatozoa with some sperm parameters and in vitro fertilization rate. METHODS: Spermatozoa were collected from 139 men, washed in PBS, fixed in methanol/glacial acetic acid (3:1), and then spread on slides. CMA3 positivity is expressed as the percentage in 200 spermatozoa. RESULTS: Percentage of CMA3 positivity showed not only a negative correlation with fertilization rate but also a significant difference between fertilizing and nonfertilizing patients. Moreover, percentage of CMA3-positive spermatozoa showed a negative correlation with count and percentage motility and a positive correlation with percentage of abnormal morphology. Percentage of CMA3 positivity also had a positive correlation with some abnormalities of head such as amorphous and macrocephaly. Ultrastructural study showed chromatin unpackaging in high CMA3-positive semen samples in comparison with low CMA3-positive semen samples. CONCLUSION: There is a close relationship among fertilization rate, sperm parameters, and CMA3 positivity and CMA3 could be considered as a useful tool for evaluation of male fertility prior to infertility treatment. (+info)
The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies.
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Human semen is heterogeneous in quality, not only between males but also within a single ejaculate. Differences in quality are evident, both when examining the classical parameters of sperm number, motility and morphology and in the integrity of the sperm nucleus. The aim of this study was to determine the efficiency of the PureSperm((R)), Percoll((R)) and swim-up preparation techniques to eliminate spermatozoa with nuclear anomalies. Semen samples were collected, washed and one part of the semen spread on a slide, the remainder was prepared using the swim-up, PureSperm((R)) or Percoll((R)) techniques. Spermatozoa from different fractions were fixed on slides and assessed. Sperm samples (n) from different men were stained using the chromomycin A(3) (CMA(3)) fluorochrome, which indirectly demonstrates a decreased presence of protamine (n = 31 for swim-up; n = 45 for PureSperm((R)); n = 39 for Percoll((R))). Spermatozoa prepared using PureSperm((R)) (n = 35) and Percoll((R)) (n = 37) were also examined for the presence of endogenous DNA nicks. Good quality spermatozoa should not possess DNA nicks and not stain (i.e. fluoresce) with CMA(3). When prepared using the swim-up technique the spermatozoa recovered showed no significant improvement with the CMA(3) staining. When spermatozoa were prepared using the PureSperm((R)) and Percoll((R)) techniques, a significant (P < 0.001) decrease in both CMA(3) positivity and DNA strand breakage was observed. These results indicate that both the PureSperm((R)) and Percoll((R)) techniques can enrich the sperm population by separating out those with nicked DNA and with poorly condensed chromatin. (+info)
Evaluation of complexation of metal-mediated DNA-binding drugs to oligonucleotides via electrospray ionization mass spectrometry.
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The interactions of self-complementary oligonucleotides with a group of metal-mediated DNA-binding drugs, including chromomycin A(3), mithramycin and the novel compound UK-1, were examined via electrospray ionization quadrupole ion trap mass spectrometry. Both chromomycin and mithramycin were shown to bind preferentially to GC-rich oligonucleotide duplexes in a 2:1 drug:metal ratio, while UK-1 was shown to bind in a 1:1 drug:metal stoichiometric ratio without a strong sequence preference. These trends were observed in the presence of Co(2+), Ni(2+) and Zn(2+), with the exception that chromomycin-Zn(2+) complexes were not readily observed. The binding stoichiometries as well as the sequence specificities are in agreement with literature reports for solution studies. Binding selectivities and stabilities of the complexes were also probed using electrospray ionization mass spectrometry. Both of the GC-rich oligomers 5'-GCGCGC-3' and 5'-GCGCATGCGC-3' exhibited a binding preference for chromomycin over mithramycin in the presence of Co(2+) and Ni(2+). Energy-variable collisionally activated dissociation of the complexes was employed to determine the stabilities of the complexes. The relative metal-dependent binding energies were Ni(2+) > Zn(2+) > Co(2+) for UK-1-oligomer complexes and Ni(2+) > Co(2+) for both mithramycin and chromomycin complexes. (+info)
Homolog pairing and two kinds of bouquets in the meiotic prophase of rye, Secale cereale.
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Chromosome configurations and structures during meiotic prophase were investigated by staining large repeated DNA sequences localized in the subtelomeric regions of all the chromosomes in rye, Secale cereale, in order to clarify when and how homolog pairing and bouquet formation occur. The changes of the spatial locations of chromosomes in the nucleus were investigated by the use of laser confocal microscopy, together with the surface-spreading method of silver nitrate staining to detect the formation of the synaptonemal complex. Homolog pairing in which homologs of four chromatids of a pair of homologs were coaligned in parallel but remained distinctly separate was microscopically detected for the first time in the present study. Homolog pairing showed the following characteristics: (1) it occurred at the leptotene-zygotene transition stage, prior to the formation of nodules and the synaptonemal complex; (2) the chromatin structure of chromosomes was in a state of decondensation; (3) it required no telomere clustering. These data suggest that homolog pairing represents a structure that indicates incipient recombination. After the homolog pairing stage, two kinds of bouquet configuration were found in zygotene. The commonly observed type was a loose bouquet, in which the subtelomeric regions were loosely aggregated. The other type was a definite bouquet, in which almost all the subtelomeric regions were conjugated, but this type was observed only in a limited number of the meiotic prophase cells of some individuals. It was concluded that the former represents the configuration of homologous recombination and the latter that of ectopic recombination. (+info)
Crystal structure of the [Mg2+-(chromomycin A3)2]-d(TTGGCCAA)2 complex reveals GGCC binding specificity of the drug dimer chelated by a metal ion.
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The anticancer antibiotic chromomycin A3 (Chro) is a DNA minor groove binding drug belonging to the aureolic family. Chro likely exerts its activity by interfering with replication and transcription. Chro forms a dimer, mediated by a divalent metal ion, which binds to G/C-rich DNA. Herein we report the first crystal structure of Chro bound to d(TTG GCCAA)2 DNA duplex solved by multiwavelength anomalous diffraction (MAD) based on the chelated Co3+ ion. The structure of the Mg2+ complex was subsequently refined at 2.15 A resolution, which revealed two complexes of metal-coordinated dimers of Chro bound to the octamer DNA duplex in the asymmetric unit. The metal ion is octahedrally coordinated to the O1 and O9 oxygen atoms of the chromophore (CPH), and two water molecules act as the fifth and sixth ligands. The two coordinated water molecules are hydrogen bonded to O2 atoms of C5 and C13 bases. The Chro dimer binds at and significantly widens the minor groove of the GGCC sequence. The long axis of each chromophore lies along and stacks over the sugar-phosphate backbone with the two attached saccharide moieties (rings A/B and C/D/E) wrapping across the minor groove. DNA is kinked by 30 degrees and 36 degrees in the two complexes, respectively. Six G-specific hydrogen bonds between Chro and DNA provide the GGCC sequence specificity. Interestingly, DNA in concert with Chro appears to act as an effective template to catalyze the deamination of Co(NH3)6(3+), as shown by circular dichroism and crystal structure data. Our results present useful structural information for designing new anticancer drug derivatives in the future. (+info)
Biosynthesis of the antitumor chromomycin A3 in Streptomyces griseus: analysis of the gene cluster and rational design of novel chromomycin analogs.
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The biosynthetic gene cluster of the aureolic acid type antitumor drug chromomycin A3 from S. griseus subsp. griseus has been identified and characterized. It spans 43 kb and contains 36 genes involved in polyketide biosynthesis and modification, deoxysugar biosynthesis and sugar transfer, pathway regulation and resistance. The organization of the cluster clearly differs from that of the closely related mithramycin. Involvement of the cluster in chromomycin A3 biosynthesis was demonstrated by disrupting the cmmWI gene encoding a polyketide reductase involved in side chain reduction. Three novel chromomycin derivatives were obtained, named chromomycin SK, chromomycin SA, and chromomycin SDK, which show antitumor activity and differ with respect to their 3-side chains. A pathway for the biosynthesis of chromomycin A3 and its deoxysugars is proposed. (+info)
Human cervical mucus can act in vitro as a selective barrier against spermatozoa carrying fragmented DNA and chromatin structural abnormalities.
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PURPOSE: We have carried out experiments to determine if human cervical mucus can act as an in vitro selective barrier against spermatozoa morphologically normal that carry genetic structural abnormalities. METHODS: Sperm chromatin abnormalities have been evaluated by Chromomycin A3 and "endogenous" nick translation. RESULTS: The data obtained have shown that spermatozoa possessing higher levels of DNA protamination are more proficient in crossing the cervical mucus barrier. Moreover, the levels of positivity to endogenous nick translation treatment was practically zero in such spermatozoa. CONCLUSIONS: We suggest that sperm penetration of cervical mucus could be used to select sperm preparations free of fragmented DNA or chromatin structural abnormalities for assisted reproduction. (+info)
Cytogenetic studies in three Pimelodella meeki populations (Pisces, Pimelodidae) from Tibagi River basin (Brazil).
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We analyzed cytogenetically specimens of Pimelodella meeki from Tibagi River at Limoeiro (LM) and from two tributaries, Couro do Boi (CB) and Gabriel da Cunha (GC) Rivers. All specimens presented 2n=46 chromosomes, which were the karyotypes composed by 15 pairs metacentric (M) + 6 pairs submetacentric (SM) + 2 pairs subtelocentric (ST). In specimens of GC, CB, and LM, the results of analyses of the nucleolus organizer regions (NORs), done by means of AgNO3 and CMA3 staining, showed that they are identical, located in terminal position on the short arm of a SM chromosome pair, and they were observed to be a size heteromorphism in some metaphase plates. FISH with 18S rDNA probe yielded evidence for these regions but not for the size variation, indicating that they are not due to a greater number of NOR cistrons in one of the homologue chromosomes. An interesting characteristic of these regions is that they could appear divided in blocks, as evidenced by all the techniques. This work makes clear the necessity for more deeply systematic studies, because of the confused taxonomic situation of the genus Pimelodella. (+info)