chromomycin-a3 and aniline-blue

To investigate testis-specific histone 2B (TSH2B) and its gene anomalies in infertile men.. Case-control study.. Basic science laboratory.. Fertile and infertile men.. Not applicable.. 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.. 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.. 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.

Topics: Case-Control Studies; Chromomycin A3; Histones; Humans; Infertility, Male; Male; Meiosis; Protamines; Proteomics; Semen; Sperm Motility; Testis

Infertility is considered as one of the major problems associated with spinal cord injury (SCI). However, the exact underlying mechanism is still unknown. Therefore, the main objective of this experimental study was to evaluate the effect of chronic SCI on sperm parameters as well as chromatin integrity and DNA of spermatozoa aspirated from cauda epididymis of rats. Forty-five adult Wistar rats were divided into three groups - SCI, sham, and control. Following laminectomy, SCI was induced onto exposed dura matter (T10). The sham group underwent laminectomy of T10 only, while the control rats were not exposed to any type of injury or medication. The cauda epididymal sperms were aspirated after 8 weeks for analysis of sperm parameters and sperm chromatin integrity with aniline blue (AB), chromomycin A3 (CMA3), sodium dodecyl sulphate (SDS), and acridine orange (AO) tests. The sperm progressive motility and normal morphology of SCI rats were significantly changed when compared with other groups (p < 0.05). In addition, AB as well as CMA3 tests were insignificantly increased in the SCI group when compared with the sham and control groups. However, SDS and AO tests were significantly changed in SCI samples when compared with the sham and control groups (p < 0.001). The results showed that chronic SCI in rat disturbs sperm parameters as well as nuclear maturity and DNA integrity of sperms. Therefore, sperm chromatin structure is compromised in SCI animals as revealed by chromatin structural probes. These alterations may reduce the fertility potential of the male gamete following SCI.

Topics: Acridine Orange; Aniline Compounds; Animals; Chromatin; Chromomycin A3; Chronic Disease; DNA Damage; Epididymis; Fluorescent Dyes; Male; Rats; Rats, Wistar; Sodium Dodecyl Sulfate; Sperm Motility; Spermatozoa; Spinal Cord Injuries

The successful implementation of ICSI has provided a unique means of allowing couples suffering from severe male infertility to achieve their reproductive goals. However, despite the great therapeutic advantages of the technique, ICSI often provides solutions to clinicians in the absence of an aetiological or pathophysiological diagnosis. The development of a sequential diagnostic schedule for patients consulting for fertility disturbances would be an ideal method of approach. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology and chromatin staining among fertile and subfertile men. Both chromomycin A3 (CMA3) and acidic aniline blue (AAB) were employed to record chromatin packaging quality among 58 men visiting the andrology laboratory. Intra- and interassay variations were initially recorded for fertile sperm donors. The coefficients of variation (CV) for all intra- and inter-assay assessments were < 12%. Chromatin packaging was significantly and negatively correlated with normal sperm morphology, namely r = 0.40 (P = 0.001) and r = 0.33 (P = 0.001) for CMA3 and AAB, respectively. Receiver operator characteristics illustrated sensitivity and specificity values of 75% and 82% for CMA3 and 60% and 91% for AAB, respectively. Significantly different CMA3 and AAB staining was recorded among men with severe teratozoospermia (< 4% normal forms) when compared with normozoospermic men (> 14% normal forms), namely 49% vs. 29% for CMA3 and 51% vs. 26% for AAB staining, respectively. Chromatin packaging assessments should be a valuable addition to the sequential diagnostic programme in an assisted reproduction arena.

Topics: Aniline Compounds; Cell Nucleus; Chromatin; Chromomycin A3; Fluorescent Dyes; Humans; Male; ROC Curve; Sensitivity and Specificity; Spermatozoa