Sperm Chromatin Status and DNA Fragmentation in Mouse Species with Divergent Mating Systems.

Sperm DNA integrity and chromatin status serve as pivotal indicators of sperm quality, given their intricate link to sperm function, embryo development, and overall fertility. Defects in chromatin compaction, which are often associated with compromised protamine content, can lead to damaged DNA strands. In this study, the chromatin status and possible correlation with DNA damage was assessed in males of three mouse species: Mus musculus, M. spretus, and M. spicilegus. We employed various staining methods, including aniline blue, methylene blue (Diff-Quik), toluidine blue, and chromomycin A3, to assess chromatin compaction in cauda epididymal sperm. Samples were also analyzed by the sperm chromatin structure assay (SCSA) to estimate DNA fragmentation (%tDFI, %HDS). Analyses were carried out on freshly collected sperm and cells incubated for 3 h in a HEPES-buffered modified Tyrode's medium simulating conditions of the female reproductive tract. Notably, the analysis of chromatin status yielded minimal abnormal values across all three species employing diverse methodologies. SCSA analyses revealed distinct variations in %tDFI between species. Following sperm incubation, the percentages of sperm stained with methylene blue exhibited differences among the species and were significantly correlated to the DNA fragmentation index. HDS demonstrated correlations with the percentages of sperm stained by aniline blue, methylene blue, and chromomycin A3. Overall, chromatin compaction was high across all species, with limited differences among them. The relationship between chromatin status and DNA integrity appeared to be related to levels of sperm competition among species.

Chromosome Analysis and Sorting Using Conventional Flow Cytometers.

The fluorescent dyes Hoechst (HO) and Chromomycin A3 (CA3) are commonly used for bivariate flow karyotyping to distinguish individual chromosomes from one another based on differences in base composition and DNA content. However, analysis of chromosomes using this fluorescent dye combination requires a flow cytometer equipped with lasers of specific wavelengths and higher power than is typical of conventional flow cytometers. This unit presents a chromosome staining technique with a dye combination of DAPI and propidium iodide (PI). Chromosomes stained using this dye combination can be analyzed on conventional flow cytometers equipped with a typical configuration of lasers and optics. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cell culture and metaphase harvest of suspension cell line Alternate Protocol 1: Cell culture and metaphase harvest of adherent cell line Basic Protocol 2: Preparation of chromosome suspension using polyamine isolation buffer Basic Protocol 3: Staining chromosomes with DAPI and propidium iodide Alternate Protocol 2: Staining chromosomes with Hoechst and Chromomycin A3 Basic Protocol 4: Bivariate flow karyotyping on a cell analyzer Basic Protocol 5: Bivariate flow karyotyping on a cell sorter Basic Protocol 6: Purification of flow-sorted chromosomes.

Proteomic and genetic dissection of testis-specific histone 2B in infertile men reveals its contribution to meiosis and sperm motility.

OBJECTIVE: To investigate testis-specific histone 2B (TSH2B) and its gene anomalies in infertile men. DESIGN: Case-control study. SETTING: Basic science laboratory. PATIENT(S): Fertile and infertile men. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): The histone and protamine status of sperm was studied by aniline blue and chromomycin A3 staining, respectively. Testis-specific histone 2B, total H2B, and phosphorylated TSH2B (pTSH2B) were estimated by Western blot analysis. The frequency of genetic polymorphisms and rare variants in H2BC1 was studied by Sanger sequencing. Phosphosites on TSH2B in sperm were identified by reverse-phase high-performance liquid chromatography purification of TSH2B followed by mass spectrometric analysis. RESULT(S): Aniline blue and chromomycin A3 staining revealed significantly higher histone retention and low protamine in sperm of infertile men. Sperm TSH2B and total H2B levels were significantly lower in oligozoospermic and oligoasthenozoospermic men (in both groups). The TSH2B levels were comparable in asthenozoospermic men; however, the pTSH2B level was significantly low. The H2BC1 gene sequencing identified 6 variants, of which 2 are rare variants (rs368672899 and rs544942090) and 4 (rs4711096, rs4712959, rs4712960 and rs4712961) are single nucleotide polymorphisms. Minor allele frequency of 5'-untranslated region variant rs4711096 was significantly lower in infertile men (OR = 0.65). The rare nonsynonymous variant, rs368672899, p.Ser5Pro was seen in 1 oligoasthenoteratozoospermic individual. Interestingly, mass spectrometric analysis identified a site on TSH2B to bear a phosphate group in the sperm of fertile men. CONCLUSION(S): Our study reveals a defect in the replacement of somatic histones with testis-specific variants in infertile men. Chromatin compaction positively correlates with sperm motility, which is suggestive of its utility in diagnostic semen analysis of infertile individuals. Our observations with TSH2B and its cognate gene in sperm of infertile men indicate an essential role for TSH2B in meiosis and its phosphorylation in sperm motility, respectively.

Chromosomal Instability and Origin of B Chromosomes in the Amazonian Glass Tetra Moenkhausia oligolepis (Günther, 1864) (Characiformes, Characidae).

B chromosomes occur in different species of the small characid fishes of the genus Moenkhausia. These supernumerary elements, that do not recombine with chromosomes of the standard A complement and follow their own evolutionary mechanism vary in number, morphology, and distribution. Here, we show karyotypic data of individuals of 2 populations of Moenkhausia oligolepis of the Brazilian Amazon (Pedro Correia and Taboquinha streams, Tocantins river basin), both with a diploid number of 50 chromosomes and karyotypic formula of 10m + 32sm + 8a. In addition to the normal complement, we also observed the occurrence of B chromosomes in the 2 populations with intra- and interindividual variation ranging from 0 to 10 Bs, independent of sex. The C-banding pattern evidenced heterochromatic blocks located mainly in the pericentromeric region of the chromosomes, while the B chromosomes appeared euchromatic. Silver-stained nucleolus organizer regions were identified in multiples sites, and some of these blocks were positive when stained with chromomycin A3. The karyotype analysis and the application of whole-chromosome painting in populations of M. oligolepis reinforce the conservation of the basal diploid number for the genus, as well as the evolutionary tendency in these fishes to carry B chromosomes. Both populations turned out to be in different stages of stability and expansion of their B chromosomes. We further suggest that the origin of these chromosomes is due to the formation of isochromosomes. Here, we identified a pair of complement A chromosomes involved in this process.

Nuclear heterogeneity is prevalent in high-quality fractionated human sperm cells typically used for assisted conception.

STUDY QUESTION: What is the nuclear heterogeneity of high-density purified human spermatozoa typically used for IVF purposes. SUMMARY ANSWER: The data show that while density gradient separation has improved the overall sperm population, there is still a large degree of nuclear heterogeneity within these cells. WHAT IS KNOWN ALREADY: Chromomycin A3 (CMA3) is an important DNA binding fluorochrome for the assessment of male-factor fertility. It is typically used to predict IVF outcomes on entire sperm ejaculates with very high receiver operating characteristic. Here we used CMA3 to characterise typical populations of human spermatozoa that would be used for IVF purposes after density gradient separation. STUDY DESIGN, SIZE, DURATION: We compared the intensity of CMA3 binding within high-dense sperm populations obtained from men. Binding heterogeneity was confirmed through fluorescence microscopy and FACS analysis independently. We also looked at CMA3 staining directly with head morphology in this sperm population. Finally, we looked at electron micrographs of nuclear heterogeneity (vacuoles, chromatin compaction) of spermatozoa following density gradient sorting of CMA3-stained cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used sperm donors who had fathered one or more children. Semen was collected after 2 days abstinence and purified over Percoll gradients. Only the high-quality spermatozoa, the same used for assisted conception, were then used. Cells were stained with CMA3 and sorted using FACS. Following this, electron micrographs were used to assess nuclear heterogeneity of CMA3-dependent sorted spermatozoa. MAIN RESULTS AND THE ROLE OF CHANCE: CMA3 staining occurs within morphologically normal as well as abnormal spermatozoa. High-intensity CMA3-stained sperm possessed large vacuoles that were not seen in the low-CMA3 population. In addition, the high-CMA3 stained cells possess higher amounts of nuclear granulation. LIMITATIONS, REASONS FOR CAUTION: The present study only describes the issues within the chromatin of these cells and does not suggest an alternate selection technique. WIDER IMPLICATIONS OF THE FINDINGS: CMA3 is one of the better reported prognostic assays in predicting pregnancy outcomes, especially in cases where the male is at fault. However, it is clear that even in fractionated populations of human spermatozoa, there are sperm cells that are morphologically normal yet possess high levels of CMA3 staining and chromatin granulation. The implication of this is that the embryologist, whom selects on the basis of sperm morphology, may choose a cell with poor chromatin, which may lead to poor embryo outcomes. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by the National Health and Medical Research council, APP1118943. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Sperm chromatin condensation and single- and double-stranded DNA damage as important parameters to define male factor related recurrent miscarriage.

The aim of the present work is to characterize the relationship between sperm protamine deficiency and single- and double-stranded DNA damage and to assess the diagnostic potential of chromomycin A3 (CMA3). For that purpose, semen samples from 90 human males with different clinical features were included (fertile donors, patients with recurrent pregnancy loss [RPL], and infertile patients). DNA condensation was analyzed by CMA3 and different types of DNA fragmentation were analyzed through the comet assay. A positive correlation between DNA condensation and single-stranded DNA fragmentation was found (Rs = .456; p = .05). CMA3 presented differences between fertile donors and all other groups (p < .001). Interestingly, patients with RPL, who were able to achieve a pregnancy, and infertile patients showed similar values of CMA3 (p > .05). Receiver operating characteristic curves and the profiles obtained by the combination of Comet assays and CMA3 indicate that the CMA3 test may be an interesting approach to distinguish those subjects with higher pregnancy loss risk from fertile donors (CMA3 area under the curve 0.928, with a confidence interval of 0.849-1.000). The present work shows that DNA condensation is related to oxidative damage, which affects mainly protamine-rich regions. The profiles observed in different clinical groups showed that CMA3 might be useful for the diagnosis of RPL risk when combined with Comet assays.

Chromomycin A3 suppresses cholangiocarcinoma growth by induction of S phase cell cycle arrest and suppression of Sp1­related anti­apoptotic proteins.

Cholangiocarcinoma (CCA) is a cancer of biliary epithelium. Late diagnosis and resistance to conventional chemotherapy are the major obstacles in CCA treatment. Increased expression of anti­apoptotic proteins are observed in CCA, which might confer chemoresistance. Thus, modulations of anti­apoptotic proteins leading to apoptotic induction is the focus of this study. Chromomycin A3 (CMA3), an anthraquinone glycoside­mithramycin A analog, was selected. CMA3 strongly binds to GC­rich regions in DNA, where specificity protein 1 (Sp1), a common transcription factor of apoptosis­related proteins, is preferentially bounded. The effects of CMA3 on anti­proliferation, cell cycle arrest and apoptosis induction in CCA cells were demonstrated by MTT assay, flow cytometry and western blot analysis. The results showed CMA3 suppressed cell proliferation in vitro in the nM range. At low doses, CMA3 inhibited cell cycle progression at S phase, while it promoted caspase­dependent apoptosis at higher doses. CMA3 induced effects of apoptosis were through the suppression of Sp1­related anti­apoptotic proteins, FADD­like IL­1ß­converting enzyme­inhibitory protein, myeloid cell leukemia­1, X­linked inhibitor of apoptosis protein, cellular inhibitor of apoptosis and survivin. The anti­CCA effects of CMA3 were confirmed in the xenograft mouse model. CMA3 retarded xenograft tumor growth. Taken together, CMA3 induced apoptosis in CCA cells by diminishing the Sp1­related anti­apoptotic proteins is demonstrated. CMA3 might be useful as a chemosensitizing agent.

Is there any relationship between human sperm parameters and protamine deficiency in different groups of infertile men?

OBJECTIVE: Abnormality in Histone-Protamine replacements has been indicated to cause sperm DNA damage and infertility. The aim of the present study was to investigate the relationships between sperm parameters in oligospermia, asthenospermia, and teratospermia with protamine deficiency in infertile men. MATERIAL AND METHOD: In this case-control study, we had three experimental groups including oligospermia (n=100), asthenospermia (n=100), and teratospermia (n=100) as well as normospermia (n=100) as controls. Sperm analyses were performed according to the recommendations of the World Health Organization (WHO, 2010) and sperm chromatin quality was assessed using Chromomycin A3 (CMA3) staining for each sample. RESULTS: The comparison of the data between groups indicated that the percentage of spermatozoa with protamine deficiency was significantly different in patients with oligospermia, asthenospermia, and teratospermia when compared with control ones. However, there was no significant correlation between sperm nuclear protamine deficiency and their parameters of the men with teratospermia using CMA3 test. Regarding the oligospermia and asthenospermia semen samples, the findings showed the negative correlations between the sperm nuclear protamine deficiency and progressive motility as well as immobility (p<0.001). CONCLUSION: The higher proportion of spermatozoa with abnormal chromatin packaging was observed in asthenospermic samples than those from other experimental groups as well as controls. It seems that normal morphology cannot have a valuable predictive value for good chromatin quality of spermatozoa, as much as normal motility characteristics, since samples with high mobility rates often have lower protamine deficiencies. The findings may provide a supportable promoting the future wider clinical application of chromatin/DNA integrity testing along with the semen analysis in male infertility.

Polymorphisms of sperm protamine genes and CMA3 staining in infertile men with varicocele.

OBJECTIVES: The aim of this study was to evaluate polymorphisms of sperm protamine genes and their effects on the result of CMA3 staining in varicocele men. MATERIAL AND METHODS: In a case control study, 128 patients with male infertility due to varicocele and 128 controls were recruited. Polymorphisms of PRM1 and PRM2 genes in extracted DNA samples were assessed by PCR-SSCP and sequencing. Protamine deficiency was also indirectly estimated by CMA3 staining. RESULT: Nine different variants including six variants in PRM1 gene and three variants in PRM2 gene were found among varicocele patients. The results showed that sperm count, motility and morphology were significantly different between control group without gene variations and varicocele group who had several variations in their protamine genes (P<0.05). CONCLUSION: Therefore, PRM1 and PRM2 variations in varicocele patients are associated with the production of spermatozoa with more protamine deficiency and this is one of the possible causes of infertility due to varicocele.

Evaluation of sperm DNA fragmentation and chromatin structure in infertile men with immotile short-tail sperm defect.

Teratozoospermia is characterised by the presence of spermatozoa with abnormal morphology. One of the morphological disorders that lead to male infertility is immotile short-tail sperm (ISTS) defect. In this study, we evaluated the levels of chromatin packing and DNA fragmentation in patients with immotile short-tail sperm defect. Semen samples were obtained from 31 infertile men with ISTS as case group and 31 normozoospermic men as a control group. Protamine status was evaluated using chromomycin A3 (CMA3) staining and sperm DNA fragmentation assessed by sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL). The percentage of positive CMA3 spermatozoa was significantly higher in patients' samples (22.6 ± 6.9) compared with controls (16.3 ± 4.2) (p < .05) and also mean (±SD) of sperm DNA fragmentation was significantly higher in patients compared with controls, as measured by TUNEL assay (10.45 ± 4.60 vs. 7.03 ± 2.86, p < .05) and SCSA (24.80 ± 13.1 vs. 15.2 ± 7.2, p < .05). According to our study, sperm DNA fragmentation and chromatin packing abnormality are significantly higher in the ISTS samples compared with normal samples. A possible explanation for this relationship is that sperm chromatin condensation and sperm flagellum formation occur during the same phase of spermatogenesis.

Polymorphic G:G mismatches act as hotspots for inducing right-handed Z DNA by DNA intercalation.

DNA mismatches are highly polymorphic and dynamic in nature, albeit poorly characterized structurally. We utilized the antitumour antibiotic CoII(Chro)2 (Chro = chromomycin A3) to stabilize the palindromic duplex d(TTGGCGAA) DNA with two G:G mismatches, allowing X-ray crystallography-based monitoring of mismatch polymorphism. For the first time, the unusual geometry of several G:G mismatches including syn-syn, water mediated anti-syn and syn-syn-like conformations can be simultaneously observed in the crystal structure. The G:G mismatch sites of the d(TTGGCGAA) duplex can also act as a hotspot for the formation of alternative DNA structures with a GC/GA-5' intercalation site for binding by the GC-selective intercalator actinomycin D (ActiD). Direct intercalation of two ActiD molecules to G:G mismatch sites causes DNA rearrangements, resulting in backbone distortion to form right-handed Z-DNA structures with a single-step sharp kink. Our study provides insights on intercalators-mismatch DNA interactions and a rationale for mismatch interrogation and detection via DNA intercalation.

CoII(Chromomycin)2 Complex Induces a Conformational Change of CCG Repeats from i-Motif to Base-Extruded DNA Duplex.

We have reported the propensity of a DNA sequence containing CCG repeats to form a stable i-motif tetraplex structure in the absence of ligands. Here we show that an i-motif DNA sequence may transition to a base-extruded duplex structure with a GGCC tetranucleotide tract when bound to the (CoII)-mediated dimer of chromomycin A3, CoII(Chro)2. Biophysical experiments reveal that CCG trinucleotide repeats provide favorable binding sites for CoII(Chro)2. In addition, water hydration and divalent metal ion (CoII) interactions also play a crucial role in the stabilization of CCG trinucleotide repeats (TNRs). Our data furnish useful structural information for the design of novel therapeutic strategies to treat neurological diseases caused by repeat expansions.

Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg2+, which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat.

Sperm chromatin and DNA integrity, methyltransferase mRNA levels, and global DNA methylation in oligoasthenoteratozoospermia / 대한생식의학회지

OBJECTIVE: To investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men. METHODS: Semen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values < 0.05 were considered to indicate statistical significance. RESULTS: Significantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression. CONCLUSION: Oligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.

Evaluation of sperm protamine deficiency and apoptosis in infertile men with idiopathic teratozoospermia / 대한생식의학회지

OBJECTIVE: Sperm morphology plays an important role in infertility, especially in cases of defects in the heads of spermatozoa. Tapered-head or elongated-head spermatozoa are examples of morphological abnormalities. The aim of this study was to compare the semen parameters, levels of protamine deficiency, and frequency of apoptosis between patients with normozoospermia and those with teratozoospermia with tapered-head spermatozoa. METHODS: Fifty-two semen samples (27 patients with tapered-head sperm and 25 fertile men) were collected and semen analysis was performed according to the World Health Organization criteria for each sample. Protamine deficiency and the percentage of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3) staining and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assays, respectively. RESULTS: Sperm concentration, motility, and normal morphology in the tapered-head spermatozoa (cases) were significantly lower than in the normozoospermic samples (controls). CMA3-reactive spermatozoa (CMA3+) in the case group were more common than in the controls. Apoptotic spermatozoa (TUNEL-positive) were significantly more common in the cases than in the controls. CONCLUSION: This analysis showed that tapered-head spermatozoa contained abnormal chromatin packaging and exhibited a high rate of apoptosis, which can be considered to be an important reason for the impaired fertility potential in teratozoospermic patients with tapered-head spermatozoa.

Absence of Peroxiredoxin 6 Amplifies the Effect of Oxidant Stress on Mobility and SCSA/CMA3 Defined Chromatin Quality and Impairs Fertilizing Ability of Mouse Spermatozoa.

Oxidative stress, the imbalance between reactive oxygen species production and antioxidant defenses, is associated with male infertility. Peroxiredoxins (PRDXs) are antioxidant enzymes with a wide distribution in spermatozoa. PRDX6 is highly abundant and located in all subcellular compartments of the spermatozoon. Infertile men have lower levels of sperm PRDX6 associated with low sperm motility and high DNA damage. In order to better understand the role of PRDX6 in male reproduction, the aim of this study was to elucidate the impact of the lack of PRDX6 on male mouse fertility. Spermatozoa lacking PRDX6 showed significantly increased levels of cellular oxidative damage evidenced by high levels of lipid peroxidation, 8-hydroxy-deoxyguanosine (DNA oxidation), and protein oxidation (S-glutathionylation and carbonylation), lower sperm chromatin quality (high DNA fragmentation and low DNA compaction, due to low levels of protamination and a high percentage of free thiols), along with decreased sperm motility and impairment of capacitation as compared with wild-type (WT) spermatozoa. These manifestations of damage are exacerbated by tert-butyl hydroperoxide treatment in vivo. While WT males partially recovered the quality of their spermatozoa (in terms of motility and sperm DNA integrity), Prdx6(-/-) males showed higher levels of sperm damage (lower motility and chromatin integrity) 6 mo after the end of treatment. In conclusion, Prdx6(-/-) males are more vulnerable to oxidative stress than WT males, resulting in impairment of sperm quality and ability to fertilize the oocyte, compatible with the subfertility phenotype observed in these knockout mice.

Dietary supplementation with astaxanthin may ameliorate sperm parameters and DNA integrity in streptozotocin-induced diabetic rats / 대한생식의학회지

OBJECTIVE: Diabetes mellitus (DM) is known to cause many systemic complications as well as male infertility. Astaxanthin (ASTX) is a powerful antioxidant that is involved in a variety of biologically active processes, including those with anti-diabetes effects. The present study investigates the effect of ASTX on the spermatozoa function in streptozotocin (STZ)-induced diabetic rats. METHODS: We divided 30 adult rats into three groups (10 rats per group), with a control group that received corn oil mixed with chow. DM was induced by intra-peritoneal injection of STZ. Eight weeks after the STZ injection, half of the diabetic animals were used as diabetic controls, and the rest were treated with ASTX for 56 days. Then the parameters and chromatin integrity of the epididymal sperm were analyzed using chromomycin A3, toluidine blue (TB), and acridine orange (AO) staining. RESULTS: The count, viability, and motility of the epididymal sperm were decreased significantly in the STZ group in comparison with the control group (count and viability, p<0.001; motility, p<0.001;0.01). ASTX increased normal morphology and viable spermatozoa compared to the STZ group (morphology, p=0.001; viability, p<0.001;0.05). The percentage of abnormal chromatins in TB and AO staining was higher in the STZ group compared to the control group (p<0.001;0.001). The mean percentage of TB and AO positive spermatozoa in STZ rats was significantly lower in the STZ+ASTX group (TB, p=0.001; AO, p<0.001;0.05). CONCLUSION: This study observed that in vivo ASTX treatment partially attenuates some detrimental effect of diabetes. Conversely, ASTX improved sperm viability, normal morphology, and DNA integrity.

Karyotype characterization and evolution in South American species of Lathyrus (Notolathyrus, Leguminosae) evidenced by heterochromatin and rDNA mapping.

Notolathyrus is a section of South American endemic species of the genus Lathyrus. The origin, phylogenetic relationship and delimitation of some species are still controversial. The present study provides an exhaustive analysis of the karyotypes of approximately half (10) of the species recognized for section Notolathyrus and four outgroups (sections Lathyrus and Orobus) by cytogenetic mapping of heterochromatic bands and 45S and 5S rDNA loci. The bulk of the parameters analyzed here generated markers to identify most of the chromosomes in the complements of the analyzed species. Chromosome banding showed interspecific variation in the amount and distribution of heterochromatin, and together with the distribution of rDNA loci, allowed the characterization of all the species studied here. Additionally, some of the chromosome parameters described (st chromosomes and the 45S rDNA loci) constitute the first diagnostic characters for the Notolathyrus section. Evolutionary, chromosome data revealed that the South American species are a homogeneous group supporting the monophyly of the section. Variation in the amount of heterochromatin was not directly related to the variation in DNA content of the Notolathyrus species. However, the correlation observed between the amount of heterochromatin and some geographical and bioclimatic variables suggest that the variation in the heterochromatic fraction should have an adaptive value.

Correlation between sperm DNA fragmentation index and CMA3 positive spermatozoa in globozoospermic patients.

The absence of the acrosome causes the situation which is called globozoospermia. There are a few studies, mostly as case reports, about correlation between levels of sperm DNA damage in patients with total round-headed spermatozoa. We investigated this correlation as well as CMA3 positive spermatozoa in 20 globozoospermic men (with more than 90% round-headed spermatozoa) attending to Royan Institute. Semen samples divided into three parts to semen analysis, to measure DNA fragmentation index (DFI) using sperm chromatin structure assay (SCSA) and to detect CMA3(+) sperm cells by chromomycin A3 staining and fluorescent microscopy. Our results showed that there were significant differences in sperm concentration, total sperm motility, and normal morphology between patients and controls group (p < 0.001). Moreover, the average of DFI and CMA3 positive spermatozoa in patients group significantly increases compared with control group (p < 0.001). A significant correlation between DFI and CMA3(+) in total population was also detected in patients group (r = 0.45, p = 0.046). To our knowledge, this is the largest study about correlation between DNA damage levels and CMA3 positive spermatozoa with round head sperm cells in total globozoospermic men. It seems that the increase in DNA damage may be because of defective sperm DNA compaction, as we detected CMA3 positive sperm cells in these patients.

Chromomycin A2 induces autophagy in melanoma cells.

The present study highlights the biological effects of chromomycin A2 toward metastatic melanoma cells in culture. Besides chromomycin A2, chromomycin A3 and demethylchromomycin A2 were also identified from the extract derived from Streptomyces sp., recovered from Paracuru Beach, located in the northeast region of Brazil. The cytotoxic activity of chromomycin A2 was evaluated across a panel of human tumor cell lines, which found IC50 values in the nM-range for exposures of 48 and 72 h. MALME-3M, a metastatic melanoma cell line, showed the highest sensitivity to chromomycin A2 after 48h incubation, and was chosen as a model to investigate this potent cytotoxic effect. Treatment with chromomycin A2 at 30 nM reduced cell proliferation, but had no significant effect upon cell viability. Additionally, chromomycin A2 induced accumulation of cells in G0/G1 phase of the cell cycle, with consequent reduction of S and G2/M and unbalanced expression of cyclins. Chromomycin A2 treated cells depicted several cellular fragments resembling autophagosomes and increased expression of proteins LC3-A and LC3-B. Moreover, exposure to chromomycin A2 also induced the appearance of acidic vacuolar organelles in treated cells. These features combined are suggestive of the induction of autophagy promoted by chromomycin A2, a feature not previously described for chromomycins.