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.

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.

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.

Evaluation of ubiquitin and annexin V in sperm population selected based on density gradient centrifugation and zeta potential (DGC-Zeta).

PURPOSE: Sperm that bypass natural apoptosis and the ubiquitin-proteasome system may find their way into semen. In order to avoid the insemination of such sperm during an intracytoplasmic sperm injection (ICSI) treatment, novel sperm selection procedures such as the Zeta procedure have been implemented. Therefore, the aim of this study was to evaluate extent of ubiquitination and external phosphatidylserine (EPS) in sperm populations selected by combines density gradient centrifugation (DGC) and Zeta electric potential in comparison to DGC and neat semen samples. METHODS: Semen samples were collected from 51 infertile men and divided into control, DGC and DGC-Zeta groups. Semen analysis was carried out according to World Health Organization criteria. The percentages of protamine deficiency, DNA fragmentation, EPS and ubiquitinated sperm were assessed by chromomycin A3 (CMA3), TUNEL, Annexin V, and immunostaining, respectively. RESULTS: Sperm selected by the DGC-Zeta procedure presented a lower percentage of sperm with protamine deficiency, abnormal morphology and DNA fragmentation while the percentage of annexin V and ubiquitin-positive sperm increased. CONCLUSION: The results of this study reveal that, DGC-Zeta improves the quality of the selected spermatozoa for ICSI and increases ubiquitination and EPS rates. We propose these alterations are part of the normal physiological process of capacitation.

The effects of age on DNA fragmentation, chromatin packaging and conventional semen parameters in spermatozoa of oligoasthenoteratozoospermic patients.

PURPOSE: To investigate the effects of male ageing on DNA fragmentation and chromatin packaging in the spermatozoa of oligoasthenoteratozoospermic (OAT) patients. METHODS: Sixty-one OAT patients and 49 men with proven fertility (controls) were included in the present study. DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labelling (TUNEL) assay, while chromatin packaging was assessed by chromomycin A3 (CMA3) staining. RESULTS: In the patient group, semen volume, percentage of normally shaped spermatozoa and sperm motility decreased significantly (P<0.05) with age, while sperm concentration and the percentage of TUNEL and CMA3 positive spermatozoa showed a statistically significant increase with age (P<0.05). In the control group, conventional semen parameters as well as DNA fragmentation and chromatin packaging did not show a statistically significant change with age (P>0.05). CONCLUSION: Increased age in OAT patients is associated with an increase in sperm concentration, DNA fragmentation and poor chromatin packaging, as well as a decline in semen volume, sperm morphology and motility.

Germinal vesicle chromatin configurations of bovine oocytes.

A common feature in the configuration of germinal vesicle (GV) chromatin in most species is that diffuse chromatin condenses into a perinucleolar ring during follicular growth; however, no such ring was observed in goat oocytes. Reports on whether bovine GV chromatin condenses into a perinucleolar ring are controversial. Besides, it is not known whether the perinucleolar ring in an oocyte represents a step toward final maturation or atresia. Changes in GV chromatin configurations during growth and maturation of bovine oocytes were studied using a new method that allows a clearer visualization of both the nucleolus and the chromatin after Hoechst and chromomycin A(3) staining. On the basis of the degree of condensation and distribution, the GV chromatin of bovine oocytes were classified into five configurations: NSN with diffuse chromatin in the whole nuclear area, N with condensed netlike chromatin, C with clumped chromatin, SN with clumped chromatin surrounding the nucleoli, and F with floccular chromatin near the nucleoli and near the nuclear envelope. Most of the oocytes were at the NSN stage in the <1.4-mm follicles, but the NSN pattern disappeared completely in follicles larger than 1.5mm. The SN pattern began to emerge in 1.5-mm follicles, and the number of SN oocytes increased while the number of oocytes with N and C configurations decreased with follicular growth. During maturation in vivo, while the number of N, C, and SN oocytes decreased, that of the F oocytes increased and reached maximum at 51h post prostaglandin injection. After that, the number of F oocytes decreased significantly because of germinal vesicle breakdown (GVBD). During maturation in vitro, GV chromatin configurations changed in a similar manner as during maturation in vivo. Fewer oocytes were at N, C, and SN stages, but more were at F and GVBD stages in the atretic than in the healthy follicles. Serum starvation slowed the F-GVBD transition of the in vitro maturing oocytes. More oocytes were of the SN or C configuration when ovaries were transported at 45-40 degrees C than at 35-30 degrees C. Most of the heated oocytes were blocked at the SN stage during in vitro maturation. It is concluded that (i) bovine GV chromatin condenses into a perinucleolar ring during follicular growth; (ii) bovine oocytes were synchronized at the F stage before GVBD; (iii) oocyte GV chromatin configurations were affected by serum starvation, high temperature, and follicular atresia.

Effect of sperm DNA damage and sperm protamine deficiency on fertilization and embryo development post-ICSI.

The aim of this study was to evaluate the effect of sperm DNA damage and protamine deficiency on fertilization and embryo development post-intracytoplasmic sperm injection (ICSI), and also to assess the effect of protamine deficiency on DNA damage. Semen samples were collected from 28 patients participating in the ICSI programme. Following sperm preparation and ICSI, the remaining processed semen samples were used to assess protamine deficiency and DNA damage employing chromomycin A3 (CMA3) staining and comet assay, respectively. Comet parameters, CMA3 percentage positivity, fertilization rate, embryo cleavage score and embryo quality score were assessed. Except for CMA3, none of the comet parameters showed significant correlation with fertilization rate. However, among comet parameters, head area and head intensity showed positive correlation with the embryo cleavage score, while comet mean intensity and head mean intensity showed a significant negative correlation with CMA3 positivity. Results of this study demonstrate that DNA fragmentation is more frequent in protamine-deficient spermatozoa. Unlike protamine deficiency, sperm DNA fragmentation does not preclude fertilization. Nonetheless, embryos derived from spermatozoa with high DNA damage have a lower potential to reach blastocyst stage.

Chromosomal mapping and molecular characterization of ribosomal RNA genes in Lebias fasciata (Teleostei, Cyprinodontidae).

Chromosome location of major (18S, 5.8S and 28S) and 5S ribosomal RNA genes (rDNAs) was examined in Lebias fasciata collected from different Italian blackish-waters, using silver (Ag)- and chromomycin A3 (CMA3)-staining and/or fluorescence in situ hybridization (FISH). Both 18S and 5S rDNA probes for FISH were obtained with polymerase chain reaction-directed cloning from genomic DNA of the examined species. Nucleolar organizer regions (NORs) containing the major rDNAs showed intraspecific polymorphism in number as detected by Ag-and CMA3-staining and FISH with the 18S rDNA probe. On the other hand, 5S rDNA loci constantly occurred on one chromosome pair and co-localized with a pair of the major rDNA loci as evidenced by two-color FISH using the 5S and 18S rDNA probes. Sequential CMA3- and Ag-NOR staining and FISH revealed apparent inactivation of some NORs. The cloned 5S rDNA was found to contain some TATA-like sequences that might play an important role in the regulation of gene expression.

Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa.

Mammalian spermiogenesis involves the replacement of histones by protamines, resulting in a highly compacted chromatin. Upon fertilization, the reverse process occurs. We have previously shown that the chromomycin A3 (CMA3) fluorochrome represents a useful tool for detecting protamine deficiency in spermatozoa. In this study we investigated CMA3 fluorochrome accessibility and the presence of endogenous nicks in maturing and fertilizing mouse sperm. Testicular sperm of stages 1-7 and 8-14 showed high positivity (> 96%) to CMA3, decreasing to 63% in stage 15-16 spermatids. In situ protamination of stage 15-16 spermatids saw an inhibition of CMA3 accessibility. Only 8% of the mature spermatozoa in the efferent ducts were CMA3-positive; this value decreased to 0% in the caput epididymidis. At fertilization, CMA, fluorescence reappears in decondensing sperm. Fluorescein isothiocyanate (FITC) fluorescence, identifying endogenous nicks, was evident in 6% of stage 1-7 spermatids, increased to 22% in stage 8-14 spermatids, and disappeared in stage 15-16 spermatids. During fertilization, endogenous nicks were not observed in decondensing sperm. We propose that 1) the presence of nicks in mouse testicular spermatids suggests that DNA cutting and ligating occurs prior to completion of protamination and 2) the absence of nicks during fertilization indicates that decondensation is not simply the reversal of the initial chromatin packaging process.

Development of enzyme immunoassay for chromomycin A3 and olivomycin using beta-D-galactosidase as a label.

An antibody specific for chromomycin A3 (CHM; byname, toyomycin) was produced in sufficiently high titer in rabbits by immunization with a CHM-bovine serum albumin conjugate, prepared using diazotized p-aminobenzoic acid as a coupling agent. CHM was also coupled with beta-D-galactosidase (EC 3.2.1.23) using diazotized m-aminobenzoic acid and was used as a tracer. With these reagents, a double-antibody enzyme immunoassay for CHM and for the CHM homologue olivomycin was developed which was highly sensitive and accurate enough to measure as little as 10 and 50 pg of each drug per assay tube, respectively. The enzyme immunoassay did not cross-react with mithramycin and drugs commonly used with CHM in combination chemotherapy for cancer treatment. Using this assay, drug levels were easily determined in blood and urine of rats following administration of CHM in a single dose of 2.0 mg/kg i.v. The sensitivity and specificity of the enzyme immunoassay for CHM and olivomycin should provide a valuable new tool for use in pharmacokinetic and toxicity studies of these drugs.