Chromomycin A3 Research Articles

Chromomycin A3 banding and chromosomal mapping of 45S and 5S ribosomal RNA genes in bottle gourd

Chromomycin A3 banding and chromosomal mapping of 45S and 5S ribosomal RNA genes in bottle gourd

PRIMER REPORTE DEL CARIOTIPO DE Colocasia oresbia: UN ESTUDIO CITOGENÉTICO COMPARATIVO ENTRE DOS VARIEDADES

Karyotypes of two Colocasia oresbia botanical varieties from Bangladesh were analyzed and compared with orcein, chromomycin A3 (CMA) and 4´-6 diamidino-2-phenylindole (DAPI). Both varieties had 2n=2x=26 chromosomes (karyotypic formula: 20m+6sm) and a pair of satellites each. Total chromosome length was 144.18±2.45 μm in C. oresbia var. oresbia and 133.02±2.75 μm in C. oresbia var. stolonifera. The karyotype of Colocasia oresbia var. oresbia is 2A whereas that of C. oresbia var. stolonifera is 1A. Six CMA and four DAPI bands were observed in C. oresbia var. oresbia and eight CMA and six DAPI bands in C. oresbia var. stolonifera. However, in these two morphologically distinct C. oresbia varieties of two different ecological zones, the same somatic chromosome number, diversification in various karyotypic parameters and CMA/DAPI-banding patterns were observed. In addition to taxonomic characters, the studied karyotype features will contribute to the characterization of these two C. oresbia varieties and to establish a base for future research. Key words: chromosome banding; CMA; DAPI; Karyotype.

Toxoplasma gondii induced sperm DNA damage on the experimentally infected rats.

Toxoplasma gondii, as an obligate protozoan parasite, can infect a wide variety of animals as well as human. As some studies have shown, toxoplasmosis decreases the fertility potency in different hosts, so there is a necessity for studies to determine the effects of T. gondii on reproductive system. Therefore, this project was aimed to investigate the effect of toxoplasmosis on the male reproductive system and sperm DNA integrity. In this experimental study, 80 Wistar male rats were divided into two groups as follows: infected group (inoculated by T.gondii tachyzoites) and control group [injected by Phosphate-buffered saline (PBS)]. Afterward, data were collected in every 10days interval. The detailed description of the sperm parameters were recorded, and then, chromatin integrity of the epididymal sperm was analyzed using Aniline blue (AB), Acridine orange (AO), Chromomycin A3 (CMA3), and Toluidine blue (TB) staining. Sperm parameters (motility, viability, count, and normal sperm) significantly decreased in the infected rats. Sperm stained by AO staining showed a higher percentage in the infected rats compared to the control group on day 70 (P = 0.03). The mean percentages of AB stained sperm on days 30 (P = 0.01) and 50 (P = 0.02) were higher than the healthy group. Also, the significant rising of the stained sperm was observed in the infected group on day 20 (P = 0.01). Sperm stained with TB in the infected group has significantly increased on days 30 to 60 [day 30 (P = 0.001), 40 (P < 0.001), 50 (P = 0.014), and 60 (P = 0.001)]. T. gondii infection leads to the diminished fertility parameters as well as the damaged DNA sperm. The parasite could temporarily interfere with the male reproductive system.

B Chromosomes and Cytogenetic Characteristics of the Common Nase Chondrostoma nasus (Linnaeus, 1758).

Supernumerary B chromosomes (Bs) are very promising structures, among others, in that they are an additional genomic compartment for evolution. In this study, we tested the presence and frequency of B chromosomes and performed the first cytogenetic examination of the common nase (Chondrostoma nasus). We investigated the individuals from two populations in the Vistula River basin, in Poland, according to the chromosomal distribution of the C-bands and silver nucleolar organizer regions (Ag-NORs), using sequential staining with AgNO3 and chromomycin A3 (CMA3). Furthermore, we analyzed the chromosomal localization of two rDNA families (45S and 5S rDNA) using fluorescence in situ hybridization (FISH) with rDNA probes. Chondrostoma nasus individuals showed a standard (A) chromosome set consisting of 2n = 50: 12 metacentric, 32 submetacentric, and 6 acrocentric chromosomes (NF = 94). Fourteen out of the 20 analyzed individuals showed 1–2 mitotically unstable submetacentric B chromosomes of different sizes. Six of them, in 14.1% of the analyzed metaphase plates, had a single, medium-sized submetacentric B (Bsm) chromosome (2n = 51) with a heterochromatic block located in its pericentromeric region. The other seven individuals possessed a Bsm (2n = 51) in 19.4% of the analyzed metaphase plates, and a second Bsm chromosome (2n = 52), the smallest in the set, in 15.5% of metaphase plates, whereas one female was characterized by both Bsm chromosomes (2n = 52) in 14.3% of the analyzed metaphase plates. AgNORs, GC-rich DNA sites, and 28S rDNA hybridization sites were observed in the short arms of two submetacentric chromosome pairs of A set. The constitutive heterochromatin was visible as C bands in the centromeric regions of almost all Chondrostoma nasus chromosomes and in the pericentromeric region of several chromosome pairs. Two 5S rDNA hybridization sites in the pericentromeric position of the largest acrocentric chromosome pair were observed, whereas two other such sites in co-localization on a smaller pair of NOR chromosomes indicate a species-specific character. The results herein broaden our knowledge in the field of B chromosome distribution and molecular cytogenetics of Chondrostoma nasus: a freshwater species from the Leuciscidae family.

Antibacterial Activity of Chromomycins from a Marine-Derived Streptomyces microflavus.

A marine-derived actinomycete (Streptomyces sp. MBTI36) exhibiting antibacterial activities was investigated in the present study. The strain was identified using genetic techniques. The 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces microflavus. Furthermore, a new chromomycin A9 (1), along with chromomycin Ap (2), chromomycin A2 (3), and chromomycin A3 (4), were isolated from the ethyl acetate extract. Their structures were determined using extensive spectroscopic methods including 1D and 2D NMR, and HRMS, as well as comparisons with previously reported data. Compounds 1–4 showed potent antibacterial activities against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). During a passage experiment, minimum inhibitory concentration (MIC) values for compounds 1–4 showed no more than a 4-fold increase from the starting MIC value, indicating that no resistance was detected over the 21 passages.

L-Carnitine reduces the negative effects of formalin on sperm parameters, chromatin condensation and apoptosis in mice: An experimental study.

BackgroundFormalin is commonly applied as an antiseptic and tissue fixative. It has reactive molecules that lead to its cytotoxic effects. According to recent studies, formalin causes a change in the testicular and sperm structure and L-carnitine (LC) acts as an antioxidant to counteract its effects.ObjectiveThis study aimed to investigate the protective effects of LC on the parameters, chromatin condensation and apoptosis of mice sperm exposed to formalin.Materials and Methods In this experimental study, 24 balb/c mice (25-40 gr,10-12 wk) were divided into three groups (n = 8/each): group I without any injections or gavage; group II, received 10 mg/ kg formalin intraperitoneally (I.P); and group III was exposed to formalin and LC, where a dose of 10 mg/kg formalin was injected I.P daily and LC the dose of 100 mg/kg was kept in a solvent solution. After 31 days, the sperm examination was performed as follows: to evaluate chromatin and DNA quality of the sperm, we applied aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3), and terminal transferase-mediated deoxy uridine triphosphate biotin end labeling (TUNEL) tests.Results Sperm parameters such as count, motility, morphology, and viability displayed a significant decrease in the formalin group. While the data exhibited a considerable augment in sperm parameters in the formalin + LC than the formalin and control groups (p 0.001), significant differences were detected between groups with respect to TB staining, TUNEL test, CMA3 test and AB staining in the formalin and formalin + LC groups.ConclusionLC can reduce the negative effects of formalin on sperm parameters, chromatin stability, and percentage of apoptosis in an animal model.

Karyotype variability of sour orange (Citrus aurantium L.) and the origin of its heteromorphic karyotypes

Sour orange (Citrus aurantium L.) is a very well-known citrus product. As sweet orange [Citrus sinensis (L.) Osbeck], it originated from the crossing between mandarin (Citrus reticulata Blanco) and pummelo [Citrus maxima (Burm.) Merril]. In order to investigate the origin and variability of its karyotype, eight cultivars of C. aurantium and one cultivar of Citrus natsudaidai Hayata, closely related to C. aurantium, were characterized with the fluorochromes CMA (chromomycin A3) and DAPI (4′-6-diamidino-2-phenylindole) and FISH (fluorescent in situ hybridization) with 5S and 35S rDNA, as well as BAC (bacterial artificial chromosomes) probes. The sour orange accessions analyzed presented three distinct karyotype formulae, but six of eight accessions had the formula 1A + 1B + 1C + 8D + 7F. Citrus natsudaidai presented the formula 2A + 1C + 6D + 9F. The accessions ‘Smooth Flat Seville’, ‘Standard’, and ‘Thornless’ presented three sites of 35S rDNA in total, in A or B chromosomes, as well as in one D. The accessions ‘Smooth Flat Seville’ and ‘Thornless’ presented always two sites of 5S rDNA, in one F and one D chromosomes. The presence of chromosome types A/35S and F/5S reinforces C. aurantium as a first-generation hybrid derived from C. maxima, while a D/5S-35S chromosome corroborates C. reticulata as a second parent. However, several chromosome changes occurred after the hybridization event, which can be partly explained by the reduction of heterochromatic blocks and partly by the occurrence of introgressions in some accessions.

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.

Study on the protective effect of hydroalcoholic Olive Leaf extract (oleuropein) on the testis and sperm parameters in adult male NMRI mice exposed to Mancozeb

This study aimed to assess the protective effects of the Olive Leaf Extract (OLE) against Mancozeb (MZB)-induced reproductive damage in male NMRI mice.In this experimental study, NMRI mice were randomly divided into 7 groups (n = 8) including 1: control, 2: MZB, 3: MZB + OLE (200 mg/kg), 4: MZB + OLE (400 mg/kg), 5: MZB + atropine (0.25 mg/kg), 6: OLE (200 mg/kg) and 7: OLE (400 mg/kg). Mice were sacrificed after 5 weeks, and blood samples were drawn to measure serum testosterone level. Sperm samples were collected and analyzed according to standard criteria. The left testis was removed to assess testicular parameters using H&E staining. The Protamine level and DNA fragmentation were determined by chromomycin A3 (CMA3) and SCD assays, respectively.Sperm viability decreased in MZB- induced groups. Also, the sperm's protamine deficiency (higher than 30%) and DNA fragmentation increased within MZB induced. There were significantly higher structural deformities of sperm's head, neck, and tail in MZB-treated groups as well (p < 0.05).Within treated groups with OLE, these sperm factors could repair defects versus MZB group. Furthermore, there were significant differences comparing sperm motility (type A, B, and D); protamine level, DNA fragmentation (SCD1, SCD3, and SCD4), and testosterone levels compared to the groups (p < 0.05). Sperm motility, seminiferous tubules diameter, the height of the germinal epithelium, and testosterone level within OLE 200 mg/kg was higher than other groups, while these factors in OLE 400 mg/kg was less than OLE 200 mg/kg.Results showed that oral administration of OLE could be useful in ameliorating MZB induced toxicity in the reproductive system of male mice within 200 mg/kg dosage; while OLE 400 mg/kg probably could exacerbate the harmful effects.

Relationship of Seminal Oxidation-Reduction Potential with Sperm DNA Integrity and pH in Idiopathic Infertile Patients

Seminal oxidative stress (OS) is one of the most promising factors to describe the causes of idiopathic male infertility. Redox balance is essential in several biological processes related to fertility, so alterations such as high reactive oxygen species (ROS) levels or low antioxidant agent levels can compromise it. MiOXSYS has been developed to evaluate the seminal static oxidation-reduction potential (sORP) and it has been proposed as an effective diagnostic biomarker. However, its relationship with parameters like sperm DNA fragmentation (SDF), chromatin compaction status or seminal pH requires further analysis, making it the object of this study. Semen and sORP analysis were performed for all samples. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) and Comet assay were used to assess SDF and chromomycin a3 (CMA3) test to assess sperm chromatin compaction. Regarding sORP measures, it was found that alkaline pH has an effect on sample reproducibility. To our knowledge, this unexpected effect has not been previously described. A statistical analysis showed that sORP correlated negatively with CMA3 positive cells and sperm motility, but not with SDF. As redox dysregulation, which occurs mainly at the testicular and epididymal level, causes chromatin compaction problems and leaves DNA exposed to damage, an excess of ROS could be counterbalanced further by a seminal supply of antioxidant molecules, explaining the negative correlation with CMA3 positive cells but no correlation with SDF. Our results show that the study of idiopathic infertility would benefit from a combined approach comprising OS analysis, SDF and chromatin compaction analysis.

Comparing the different methods of sperm chromatin assessment concerning ART outcomes.

Sperm DNA fragmentation and maturation directly interferes with reproductive efficiency. Although there are several methods for assessing sperm DNA integrity, however, many of them are laborious and require high-precision equipment in the clinics. Thus, evaluating economic and reliable methods to prepare suitable sperm for assisted reproductive technologies without DNA damage is critical. A total of 114 semen samples were collected and analyzed using computer-assisted semen analysis. The DNA fragmentation index (DFI) of all samples was evaluated by two methods of sperm chromatin dispersion (SCD) and sperm chromatin structure assay (SCSA). Besides, chromatin maturation index (CMI) was assessed by three methods including aniline blue (AB)-sperm chromatin maturation assay (SCMA), fluorescence microscopic chromomycin A3 (fmCMA3), and flow cytometric CMA3 (fcCMA3). The result showed that the DFI had no statistically significant differences (p>0.05) between SCSA (26.98%±1.28%) and SCD (27.88%±1.278%), although SCD demonstrated a strong correlation with DNA maturity (p<0.001), which had not been seen in SCSA. Besides, the CMI demonstrated significant differences (p<0.001) when assessed by AB-SCMA (14.86%±0.65%), fmCMA3 (29.18%±1.01%), and fcCMA3 (22.45%±0.62%). Among these, only the fmCMA3 showed a significant correlation with semen parameters (p<0.01) and embryo development (p<0.001). It seems that SCD and fmCMA3 were more accessible, affordable, and reliable tests for assessing DFI and CMI. It appeared these two methods may be the best choices for evaluating sperm DNA integrity in clinics.

Could high DNA stainability (HDS) be a valuable indicator of sperm nuclear integrity?

BackgroundThe Sperm Chromatin Structure Assay (SCSA®), in addition to identifying the DNA Fragmentation Index (DFI) also identifies High DNA satiability (HDS), supposed to reflect the nuclear compaction of spermatozoa. However, data on what exactly this parameter reveals, its relevance and usefulness are contradictory. In order to shed light on this situation, spermatozoa of a cohort (N = 397) of infertile men were subjected to the SCSA®, TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling) and CMA3 (Chromomycin A3) tests. In a smaller subcohort (N = 100), aniline blue (AB) and toluidine blue (TB) staining were performed in addition. The objective of this study was thus to answer the question of whether HDS is a relevant and reliable parameter to be taken into account?ResultsHDS does not appear to be a reliable indicator of nuclear immaturity because it shows a weak correlation with the CMA3, AB and TB stains. The low correlation of HDS with sperm DNA fragmentation (TUNEL and SCSA®) and DNA condensation (CMA3, AB and TB) tests suggests that these two parameters could be decoupled. Unlike DFI and TUNEL, HDS has not been shown to correlate with classic clinical situations of male infertility (asthenozoospermia, teratozoospermia or astheno-teratozoospermia).ConclusionHDS correlates poorly with most tests that focus specifically on the level of maturity of the sperm nucleus. To our knowledge, this study is the first to compare SCSA®, TUNEL, AB, TB and CMA3 assays on identical samples. It shows the potency, consistency and limitations of each test and the care that must be taken in their interpretation.

Unravelling the genetic diversity and phylogenetic relationships of Indian Capsicum through fluorescent banding

Hot chilli species of Capsicum are one of the most significant spice crops in India having several genetically distinct cultivars. The present study was targeted to upgrade chromosomal database of 15 cultivars belonging to C. annuum L., C. frutescens L. and C. chinense Jacq. EMA-based fluorochrome banding with GC-specific stain Chromomycin A3 (CMA) was elemental to reveal specialization in karyotypes that could not be traced out by Giemsa-stained preparations. CMA banding pattern was combined with karyomorphometric indices to statistically evaluate chromosomal relationship among the cultivars. The marker chromosome pairs 11 and 12 containing nucleolar CMA bands could be suggested as the evolutionary landmarks in ‘C. annuum complex’. We found direct correspondence between fruit morphotype variation and CMA banding pattern to demonstrate maximum genetic diversity within C. annuum, followed by C. frutescens while Bhut jolokia of C. chinense displayed genetic uniformity. UPGMA phenogram upheld distinct species separation, cultivar diversity and relationships within and among the species. Out of nine C. annuum cultivars, at least two cultivars showed some sort of genetic relationship with C. chinense, particularly the Ghee cultivar shares more proximity with C. chinense. The fluorescent karyotype database reliably symbolized uniqueness of Capsicum germplasm of India, especially from the NEH sector that grows Bhut jolokia. Our attempt is believed to complement genomic investigation in the popular and exotic chilli cultivars of India and interest breeders in search of alternative genetic resources.

Evidence of Interspecific Chromosomal Diversification in Rainbowfishes (Melanotaeniidae, Teleostei).

Rainbowfishes (Melanotaeniidae) are the largest monophyletic group of freshwater fishes occurring in Australia and New Guinea, with 112 species currently recognised. Despite their high taxonomic diversity, rainbowfishes remain poorly studied from a cytogenetic perspective. Using conventional (Giemsa staining, C banding, chromomycin A3 staining) and molecular (fluorescence in situ hybridisation with ribosomal DNA (rDNA) and telomeric probes) cytogenetic protocols, karyotypes and associated chromosomal characteristics of five species were examined. We covered all major lineages of this group, namely, Running River rainbowfish Melanotaenia sp., red rainbowfish Glossolepis incisus, threadfin rainbowfish Iriatherina werneri, ornate rainbowfish Rhadinocentrus ornatus, and Cairns rainbowfish Cairnsichthys rhombosomoides. All species had conserved diploid chromosome numbers 2n = 48, but karyotypes differed among species; while Melanotaenia sp., G. incisus, and I. werneri possessed karyotypes composed of exclusively subtelo/acrocentric chromosomes, the karyotype of R. ornatus displayed six pairs of submetacentric and 18 pairs of subtelo/acrocentric chromosomes, while C. rhombosomoides possessed a karyotype composed of four pairs of submetacentric and 20 pairs of subtelo/acrocentric chromosomes. No heteromorphic sex chromosomes were detected using conventional cytogenetic techniques. Our data indicate a conserved 2n in Melanotaeniidae, but morphologically variable karyotypes, rDNA sites, and heterochromatin distributions. Differences were observed especially in taxonomically divergent species, suggesting interspecies chromosome rearrangements.

Does finasteride treatment for benign prostatic hyperplasia influence sperm DNA integrity in dogs?

BackgroundBenign prostatic hyperplasia (BPH) is one of the most common reproductive disorders in both male dogs and men. Finasteride, a synthetic inhibitor of the enzyme 5α-reductase, is widely used as medical treatment. Although sperm can be affected by both BPH and finasteride treatment, the direct influence on DNA integrity remains unclear. Thus, the aim of this study was to verify the direct effect of BPH and/or finasteride treatment on DNA integrity of dog spermatozoa. A 2 × 2 factorial experiment was designed with 20 male dogs assigned to 4 experimental groups: BPH Group (n = 5), BPH-Finasteride Group (n = 5), Non-BPH Finasteride-Treated Group (n = 5) and Non-BPH Untreated Group (n = 5). Sperm evaluation was performed monthly for 60 days after the start of finasteride therapy or BPH diagnosis (D0, D30 and D60). Sperm DNA integrity was analyzed through fragmentation susceptibility (toluidine blue staining and Sperm Chromatic Structure Assay - SCSA), direct evaluation of DNA fragmentation (Sperm Chromatin Dispersion Assay - SCDA) and sperm protamination (chromomycin A3).ResultsSperm DNA integrity was not affected by finasteride treatment. However, BPH dogs had higher susceptibility to sperm DNA acid denaturation (SCSA) compared to dogs not presenting BPH, as well as lower percentage of sperm with DNA integrity (toluidine blue staining).ConclusionIn conclusion, benign prostatic hyperplasia causes post-testicular sperm DNA damage, albeit finasteride treatment itself does not directly influence sperm DNA integrity.

Effects of crocin on frozen-thawed sperm apoptosis, protamine expression and membrane lipid oxidation in Yanbian yellow cattle.

Glycerol is used as a bovine semen osmotic cryoprotectant that greatly improves the quality of frozen and thawed bovine sperm. However, high glycerol concentrations can have a toxic effect on frozen and thawed bovine sperm. Therefore, this experiment investigated the effect of replacing a portion of the glycerol in a cryoprotectant solution with crocin on the sperm apoptosis, protamine deficiency and membrane lipid oxidation of frozen and thawed Yanbian yellow cattle sperm. The experiment included a control group (6% glycerol) and four treatment groups: I (3% glycerol), II (3% glycerol +0.5mM crocin), III (3% glycerol+1mM crocin) and IV (3% glycerol+2mM crocin). Computer assisted semen analysis was used to detect sperm motility, Hoechst 33,342, propidium iodide, and JC-1 staining were used to analyse sperm viability and mitochondrial membrane potential, chromomycin A3 staining was used to detect protamine deficiency and DNA damage, flow cytometry was used for sperm membrane lipid disorder detection and analysis, and real-time quantitative RT-qPCR was used to detect the mRNA expression levels of protamine-related genes (PRM2, PRM3), sperm acrosome-associated genes (SPACA3), oxidative stress-related genes (ROMO1) and apoptosis-related genes (BCL2, BAX). Compared to the control group, replacing a portion of glycerol with 1mM crocin significantly improved sperm motility, plasma membrane integrity, membrane lipid disorders (p<.05) and viability, mitochondrial membrane potential, protamine deficiency (p<.01). The expression level of PRM2, PRM3, SPACA3 and BCL2 significantly increased (p<.05), while the expression levels of ROMO1 and BAX significantly decreased (p<.05). Accordingly, the BCL2/BAX ratio significantly increased (p<.05). In summary, the substitution of a portion of glycerol with crocin in cryoprotective solution improved the quality of Yanbian yellow cattle sperm after freezing and thawing.

Comparative analysis of tests used to assess sperm chromatin integrity and DNA fragmentation.

Male infertility has a complex etiology, and many times, the cause is unknown. While routine semen analysis provides an overview of basic semen parameters, such as sperm concentration, motility, viability and morphology, a significant overlap of these parameters has been reported in fertile and infertile men. Moreover, conventional semen parameters do not reveal the cellular or molecular mechanisms of sperm dysfunctions leading to infertility. Therefore, sperm functional parameters, including sperm chromatin integrity, are evaluated to provide information on subtle sperm defects that are not routinely identified. Incomplete or defective sperm chromatin condensation increases the susceptibility of the sperm DNA to oxidative damage or other factors. To evaluate sperm chromatin integrity, different methods with varying degrees of diagnostic and prognostic capabilities are available. Among these assays, SCSA, TUNEL and SCD assays are most commonly used. While these assays rather evaluate the DNA directly for damages, the aniline blue and chromomycin A3 stains test for the quality of chromatin condensation. Thus, this review discusses and compares different methods used to evaluate sperm chromatin integrity and condensation, and their inclusion in the routine evaluation of the male infertility.

Karyotypic variability in Calliandra sect. Androcallis (Leguminosae–Caesalpinioideae)

Calliandra is a neotropical genus composed of 139 species with distribution in North, Central and South America. In this work, a comparative cytogenetic analysis of 10 species of the Androcallis section was performed by double staining with base-specific chromomycin A3 (CMA) and 4′,6-diamidino-2-phenylindole (DAPI). The DNA content of the species was also estimated by flow cytometry. This study revealed that the species C. macrocalyx, C. riparia, C. imperialis, C. ulei, C. spinosa and C. umbellifera are diploid with 2n = 2x = 16, C. fernandesii is tetraploid with 2n = 4x = 32, C. sessilis is octaploid with 2n = 8x = 64 and C. dysantha is decaploid with 2n = 10x = 80. This result revealed that polyploidy is one of the recurrent cytogenetic events in the evolution of the genus Calliandra. As for the number of CMA+ bands, it was possible to characterize each species. No AT-rich DAPI band was located on the chromosomes of the species. The results suggest that chromosomal rearrangements and polyploidy contributed to the evolution of Calliandra species, as demonstrated by the cytotaxonomic characterization in the genus.

Effects of acrylamide in the presence of vitamin E on sperm parameters, chromatin quality, and testosterone levels in mice.

ObjectiveThe present study investigated sperm chromatin quality and testosterone levels in acrylamide-treated mice and the possible protective effects of vitamin E on the fertility potential of spermatozoa.MethodsThirty-two adult male mice were divided equally into four groups. Group 1 was the control, group 2 received acrylamide (10 mg/kg, water solution), group 3 received vitamin E (100 mg/kg, intraperitoneal), and group 4 received both acrylamide and vitamin E. After 35 days, spermatozoa from the right cauda epididymis were analyzed in terms of count, motility, morphology, and viability. Sperm DNA integrity and chromatin condensation were assessed by acridine orange (AO), aniline blue (AB), toluidine blue (TB), and chromomycin A3 (CMA3) staining.ResultsIn acrylamide-treated mice, significantly lower sperm concentration, viability, motility, and testosterone levels were found in comparison with the control and acrylamide+vitamin E groups (p<0.05). In the vitamin E group, significantly more favorable sperm parameters and testosterone levels were found than in the other groups (p<0.05). There were also significantly more spermatozoa with less condensed chromatin in the acrylamide-treated mice than in the other groups. Moreover, significantly more spermatozoa with mature nuclei (assessed by AB, CMA3, AO, and TB staining) were present in the vitamin E group than in the control and acrylamide+vitamin E groups.Conclusion This study revealed the deleterious effects of acrylamide on sperm parameters and sperm chromatin quality. Vitamin E can not only compensate for the toxic effects of acrylamide, but also improve sperm chromatin quality in mice.

Anadolu’da Yaşayan Endemik Üç Pseudophoxinus Bleeker, 1860 (Teleostei: Leuciscidae) Türünün CMA3 ve DAPI Boyalı Kromozomları

The karyotypes and other chromosomal markers of representatives of the genus Pseudophoxinus have been little studied. Therefore, this study documents chromosomal NOR phenotypes as revealed using Chromomycin A3 (CMA3) and DAPI chromosomal stainings in three species Pseudophoxinus elizavetae Bogutskaya, Küçük &amp;amp; Atalay, 2007, P. firati Bogutskaya, Küçük &amp;amp; Atalay, 2007 and P. hittitorum Freyhof &amp;amp; Özuluğ, 2010, endemic in Anatolia. Simple NOR phenotype with only two CMA3 positive signals were observed in the karyotype of P. firati whereas derived NOR phenotype with four CMA3 positive signals were observed in those of P. elizavetae and P. hittitorum, respectively. No DAPI positive signal was detected in karyotypes in all three species. This study described new NOR phenotypes, i.e. number and position of major rDNA genes, in genomes of three Pseudophoxinus species contributing thus to known diversity of NOR phenotypes in otherwise karyotypically highly conservative leuciscid fishes.