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Aim: The current sperm selection procedure for intracytoplasmic sperm injection has limited ability to detect structural and functional abnormalities of the spermatozoa. The aim of this study was to investigate whether the degree of sperm tail swelling observed during hypo-osmotic swelling test (HOST) may predict sperm quality.Materials & methods: Sixty semen samples were collected from men investigated for couple infertility. For each sample, sperm parameters, HOST and sperm chromatin status were evaluated. The relationship between the different HOST-induced tail swelling patterns (‘a’ to ‘g’) and sperm quality was evaluated.Results: The HOST significantly correlated with higher sperm motility and vitality, and with better morphology and nuclear quality. The HOST grades ‘b’ and ‘c’ were associated with better motility (p

Paternal obesity has been implicated in adult-onset metabolic disease in offspring. However, the molecular mechanisms driving these paternal effects and the developmental processes involved remain poorly understood. One underexplored possibility is the role of paternally induced effects on placenta development and function. To address this, we investigated paternal high-fat diet-induced obesity in relation to sperm histone H3 lysine 4 tri-methylation signatures, the placenta transcriptome, and cellular composition. C57BL6/J male mice were fed either a control or high-fat diet for 10 weeks beginning at 6 weeks of age. Males were timed-mated with control-fed C57BL6/J females to generate pregnancies, followed by collection of sperm, and placentas at embryonic day (E)14.5. Chromatin immunoprecipitation targeting histone H3 lysine 4 tri-methylation (H3K4me3) followed by sequencing (ChIP-seq) was performed on sperm to define obesity-associated changes in enrichment. Paternal obesity corresponded with altered sperm H3K4me3 at promoters of genes involved in metabolism and development. Notably, altered sperm H3K4me3 was also localized at placental enhancers. Bulk RNA-sequencing on placentas revealed paternal obesity-associated sex-specific changes in expression of genes involved in hypoxic processes such as angiogenesis, nutrient transport, and imprinted genes, with a subset of de-regulated genes showing changes in H3K4me3 in sperm at corresponding promoters. Paternal obesity was also linked to impaired placenta development; specifically, a deconvolution analysis revealed altered trophoblast cell lineage specification. These findings implicate paternal obesity effects on placenta development and function as one potential developmental route to offspring metabolic disease.

Numerous studies have demonstrated the detrimental effects of sperm DNA fragmentation (SDF) on reproductive outcomes, whether under natural or assisted conditions. The sperm DNA fragmentation (SDF) test has emerged as a valuable supplement in the evaluation of infertile men, and the rapid development of various SDF tests has enhanced the perception of its clinical utility. Consequently, the latest manual from the World Health Organization has determined that this test should be described as a specialized analysis (and thus applicable in many laboratories worldwide) rather than a research analysis. However, the normal reference ranges for these tests to evaluate male fertility potential remain controversial. This article addresses the pathophysiological aspects of sperm chromatin and the mechanisms underlying DNA fragmentation. The effects of this fragmentation on various reproductive outcomes are also reviewed. An analysis of the different available fragmentation tests is provided as well. Finally, we discuss preventive and therapeutic measures to reduce sperm DNA fragmentation and potential future research opportunities. [ABSTRACT FROM AUTHOR]

Sperm chromatin is distinct from somatic cell chromatin, as a result of extensive remodeling during the final stages of spermatogenesis. In this process, the majority of histones is replaced with protamines. The chromatin is consequently highly condensed and inert, which facilitates protection of the DNA. The sperm epigenomic landscape is shaped by histone retention, histone and protamine modification, DNA methylation, and RNAs. In recent years, sperm chromatin integrity and its epigenetic marks have been increasingly studied, and the constitution of sperm chromatin is steadily being uncovered. This growing body of research prompts assessment of the frequently overlooked involvement of sperm in fertility and embryonic development. Moreover, numerous endogenous and exogenous factors are known to affect sperm chromatin, which may in turn impact the reproductive success. Concerns have been raised about the effects of assisted reproductive technology (ART) on the sperm epigenome, embryonic development and offspring health. This review examines the structure and epigenetic signatures of sperm chromatin in the context of fertility and early embryonic development. Additionally, sperm chromatin evaluation and causes of aberrant integrity are outlined. Building on the knowledge discussed in the current review, future research should aim to elucidate the intricate relationship between all aspects of sperm chromatin and embryo development. This could lead to the uncovering of new targets for treating infertility, as well as the acquisition of much needed insights into the possible reciprocal association between ART and sperm chromatin integrity.

Aim: To evaluate the relationship between sperm chromatin condensation assessed by aniline blue staining and semen parameters in infertile men. Methods: Infertile men applied to our urology clinics and diagnosed with normozoospermia (n=50), asthenozoospermia (n=17), oligozoospermia (n=3), teratozoospermia (n=2) oligoasthenoteratozoospermia (OAT) (n=10) according to their semen analysis were included in the study. Semen samples were evaluated for sperm chromatin condensation by aniline blue staining. Results: The percentage of aniline-positive spermatozoa in the OAT and teratozoospermia group was found to be higher than in the normozoospermic and asthenozoospermic infertile groups (p=0.002, p=0.044 with normozoospermia group and p=0.026, p=0.007 with asthenozoospermia group, respectively). Sperm chromatin condensation was negatively correlated with sperm concentration (p=0.003, r=-0.322), total sperm count (p=0.004, r=-0.313), total progressive motile sperm count (p=0.005, r=-0.307), and normal morphology (p<0.0001, r=-0.554); and positively correlated with the percentage of immotile sperm (p=0.037, r=0.230). Conclusion: Sperm chromatin condensation was found to be different in infertile men differently diagnosed based on their semen analysis. The results of the study suggest that chromatin condensation, together with routine sperm parameters, may constitute a valuable parameter in the evaluation of male fertility. [ABSTRACT FROM AUTHOR]

Today, plastic pollution is a widespread problem in all ecosystems and has a particularly severe impact on marine ecosystems and external fertilisers such as the mussel Mytilus galloprovincialis. The present study aims to assess the toxicological reproductive health effects in this organism following exposure to two concentrations of polystyrene microplastics (PS-MPs) (0.5 and 1 μg/L), representative of conditions in the Mediterranean Sea. After exposure, the electrophoretic pattern of protamine-like (PL) proteins, the major basic protein component of Mytilus galloprovincialis sperm chromatin, was analysed. Compared to the unexposed condition, differences were observed by SDS-PAGE and an increased ability of PL to bind and protect DNA from oxidative damage was then measured, particularly for PL from mussels exposed to 1 μg/L PS-MPs. At this dose of PS-MPs, a reduced release of all PLs from the sperm nuclei was also observed, whereas the digestion by micrococcal nuclease did not show any significant differences between the exposed and the unexposed conditions. Finally, the possibility of poly(ADP)-ribosylation of the PLs was investigated. PL-II showed an increase in poly(ADP)-ribosylation after PS-MPs exposure, which may account for the difference in the ability of the PLs to bind DNA. In conclusion, while all the results might suggest a molecular mechanism of gametic plasticity occurring upon exposure of mussels to PS-MPs 1 μg/L, they also indicate that this dose of exposure could be extremely detrimental to the reproductive health of Mytilus galloprovincialis because it could prevent the release of basic nuclear proteins from the sperm DNA at fertilisation. • Microplastics cause an increase of ɣH2AX a marker of DNA double strand break. • Microplastics produce changes in Mytilus galloprovincialis PL to DNA binding. • Microplastic exposure increased poly(ADP)-ribosylation in PL-II and H1 histone. • 1 μM/mL polystyrene microplastic reduces the release of PLs from the sperm nuclei. • Microplastics have toxicological effects and a potential reproductive risk. [ABSTRACT FROM AUTHOR]

Male fertility potential depends on physical, endocrine, and genetic factors responsible for producing functional male gametes. Although the main function of the male gamete, the spermatozoon, is to deliver its genetic material to the oocyte, this premise has been modified over the past few years. It is believed that the spermatozoon provides essential factors for fertilization and pre-implantation embryo development. A viable/healthy spermatozoon has functional subcellular compartments (nucleus, acrosome, midpiece, and flagellum) due to the actions of proteins, transcripts, and epigenetic marks in the organelles present in them that have important roles in reproductive biology. Male fertility potential reflects viable spermatozoa with proper function. Therefore, new approaches to functional sperm analysis are essential. Additionally, intrinsic factors and sperm molecules constitute potential biomarkers of viable spermatozoa and male fertility. Among these factors are proteins, the genome, and coding and non-coding RNAs, such as microRNAs, that act during fertilization and early embryo development. Research has been seeking increasingly efficient tools to predict fertility and functional studies of these molecules through gene and protein expression. Thus, analytical tools are essential to identify and classify viable and functional spermatozoa, to evaluate assisted reproductive male potential. • The evaluation of sperm function could involve more than usual analyses. • The knowledge of sperm morphology and function can assist in the interpretation and prediction of male fertility. • There is a direct relationship between the male subject and the fertility. • The sperm intrinsic factors such as microRNAs confer individual features that modulate male fertility. [ABSTRACT FROM AUTHOR]

Th e purpose of this study was to observe the eff ect of diff erent thawing methods on semen parameters such as motility and morphology and sperm chromatin integrity as assessed by toluidine blue (TB) staining. A total of 20 frozen sperm straws from the same Holstein bull were used. While the 30 sec thawing protocol at 37°C, which is used for thawing frozen sperm straws, constitutes our slow thawing group (n=10), the 6 sec thawing protocol at 70°C constitutes our fast-thawing group (n=10). Th e motility, viability, morphology, plasma membrane integrity, and sperm chromatin condensation parameters of all thawed sperm were investigated. Th ere was a significant diff erence (P

STUDY QUESTION Do defects in sperm chromatin protamination and condensation have an impact on ICSI outcomes? SUMMARY ANSWER Sperm protamination is related to fertilization rates in healthy donors, and the in vitro capacity of sperm to condense their chromatin is linked to blastocyst rates, both associations being more apparent in women <33 years of age. WHAT IS KNOWN ALREADY Previous data on how sperm chromatin damage affects ICSI outcomes are inconsistent. Revealing which sperm factors influence embryo development is necessary to understand the male contribution to ICSI success and to develop novel sperm selection techniques or male-based treatments. Sperm chromatin is mainly condensed in protamines, which are cross-linked through disulphide bridges. This study aimed to determine whether sperm protamination and the integrity of disulphide bonds (condensation) are related to embryo development after ICSI. STUDY DESIGN, SIZE, DURATION The design was a retrospective study with a blind analysis of sperm chromatin. Gametes were divided into two groups: double donation (DD) cohort and single donation (SD) cohort. Samples from 45 semen donors used in 55 ICSI cycles with oocyte donors (age range 19–33 years), generating 491 embryos, were included in the DD cohort. The SD cohort consisted of samples from 34 semen donors used in 41 ICSI cycles with oocytes from healthy females (single-parent families or lesbian couples, age range 20–44 years), generating a total of 378 embryos. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Donor sperm samples from DD and SD cohorts were used for standard ICSI, and embryo development was observed by time-lapse imaging. The incidence of thiol reduction (dibromobimane, DBB) and the degree of chromatin protamination (chromomycin A3, CMA3, indicating non-protaminated regions) in sperm were determined by flow cytometry at 0 and 4 h post-thawing. MAIN RESULTS AND THE ROLE OF CHANCE Percentages ± standard deviation of CMA3 were 21.08 ± 9.09 and 35.01 ± 14.68 at 0 and 4 h post-thawing, respectively, in the DD cohort and 22.57 ± 9.48 and 35.79 ± 12.58, at 0 and 4 h post-thawing, respectively, in the SD cohort. Percentages of DBB+ were 16.57 ± 11.10 and 10.51 ± 8.40 at 0 and 4 h post-thawing (P  <   0.0001), respectively, in the DD cohort and 17.98 ± 10.19 and 12.72 ± 8.76 at 0 and 4 h post-thawing (P  <   0.0001), respectively, in the SD cohort. Female age correlated with fertilization rates, and the relation between sperm chromatin and embryo development was determined through multiple linear regression. While CMA3 was associated with fertilization rates, with no influence of female age, in the DD cohort (β1 = −1.036, P  <   0.001 for CMA3; β2 = 0.667, P  =   0.304 for female age), this was not observed in the SD cohort, where female age had a significant effect, masking the effects of CMA3 (β1 = −0.066, P  =   0.804 for CMA3; β 2 = −1.451, P  =   0.003 for female age). The in vitro capacity of sperm to condense their chromatin after 4 h of incubation was associated with blastocyst rates, independent of female age (DD cohort: β1 = −0.238, P  =   0.008 for %DBB+ variation; β2 = 0.404, P  =   0.638 for female age; SD cohort: β1 = −0.278, P =  0.010 for %DBB+ variation; β2 = −0.292, P  =   0.594 for female age). The in vitro capacity of sperm to condense their chromatin was also related to the time required for the embryo to reach blastocyst stage in the DD cohort (P  =   0.007). Finally, multiple logistic regression showed that both chromatin protamination and condensation, together with the age of the oocyte donors and the embryo recipients, had an impact on pregnancy achievement (P  <   0.01) and on live birth rates (P  <   0.01). LIMITATIONS, REASONS FOR CAUTION The main limitation was the restrictive selection of couples, which led to a relatively small sample size and could influence the observed outcomes. For this reason, and to reduce Type I error, the level of significance was set at P ≤  0.01. On the other hand, the use of cryopreserved samples could also be a limitation. WIDER IMPLICATIONS OF THE FINDINGS This research demonstrated that protamination and condensation of sperm chromatin are related to embryo development after ICSI, but female age could be a confounding factor when oocytes from older females are used. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the European Union's Horizon 2020 Research and Innovation scheme under the Marie Skłodowska-Curie grant agreement No 801342 (Tecniospring INDUSTRY; TECSPR-19-1-0003); La Marató de TV3 Foundation (214/857-202039); the Ministry of Science and Innovation, Spain (IJC2019-039615-I); the Catalan Agency for Management of University and Research Grants, Regional Government of Catalonia, Spain (2017-SGR-1229); and the Catalan Institution for Research and Advanced Studies, Spain (ICREA). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Nickel is associated with reproductive toxicity, but little is known about the molecular mechanisms of nickel-induced effects on sperm chromatin and protamine-like proteins (PLs). In the present work, we analyzed PLs from Mytilus galloprovincialis by urea-acetic acid polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and assessed their binding to DNA by Electrophoretic Mobility Shift Assay (EMSA) after exposing mussels to 5, 15, and 35 µM NiCl2 for 24 h. In addition, a time course of digestion with MNase and release of PLs from sperm nuclei by the NaCl gradient was performed. For all exposure doses, in AU-PAGE, there was an additional migrating band between PL-III and PL-IV, corresponding to a fraction of PLs in the form of peptides detected by SDS-PAGE. Alterations in DNA binding of PLs were observed by EMSA after exposure to 5 and 15 µM NiCl2, while, at all NiCl2 doses, increased accessibility of MNase to sperm chromatin was found. The latter was particularly relevant at 15 µM NiCl2, a dose at which increased release of PLII and PLIII from sperm nuclei and the highest value of nickel accumulated in the gonads were also found. Finally, at all exposure doses, there was also an increase in PARP expression, but especially at 5 µM NiCl2. A possible molecular mechanism for the toxic reproductive effects of nickel in Mytilus galloprovincialis is discussed. [ABSTRACT FROM AUTHOR]

Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote. [ABSTRACT FROM AUTHOR]

Present research aim was to identify functional tests in semen associated with DNA damage and chromatin maturity (protamination) which predict the outcome in assisted reproduction. Couples were grouped according to male partner semen parameters, into normozoospermia (NZs), severe male factor (SMF) and mild male factor (MMF). DNA fragmentation index (DFI) in spermatozoa was analysed by sperms chromatin dispersion (SCD), sperm chromatin structure assay (SCSA) and acridine orange testing (AOT). Chromomycin A3 (CMA3) and toluidine blue (TB) staining to measure sperm chromatin maturity (CM). DFI and chromatin decondensation were significantly lower in N compared to male factor categories (MMF and SMF). Aneuploidy embryos were significantly higher in couples with male factor infertility (MMF and SMF). A positive correlation was observed between fertilization rate (FR) and live birth rate (LBR) with sperm concentration, motility, vitality, normal sperm morphology and negative correlation between sperm DFI and sperm CM. No correlation was observed between embryo aneuploidy and sperm DFI or CM. Lower percentage of spermatozoa chromatin integrity are associated with low fertilization and live birth rate. Male factor infertility, due to impaired semen parameters and chromatin defects could be regarded in future as an indication of IVF/ICSI, and predictor of assisted reproductive techniques outcome. [ABSTRACT FROM AUTHOR]

The objective of this research was to evaluate the effect of sperm on chromatin stability and its relationship with the membrane integrity structural – physiological and the rate of fertilization of female sheep. Ejaculates of sperm (2 × 109 sperm·mL-1) with 70% of motility were collected using an artificial vagina (n=5, 2 years old. For this, each ram was served with fifteen female sheep (n=75), generating thus five different Groups (A, B, C, D, and E). A control Group also was considered. Sperm nuclear chromatin stability (NCS) was evaluated using the Borate Buffer (BB), Sodium Dodecyl Sulfate (SDS), and the mixture of Ethylenediaminetetraacetic acid (EDTA) + SDS. The fertilization rate was evaluated after 16-18 hours post sperm injection. Sperm concentration showed a significant difference (P0.05). A high correlation (r2=0.52) was observed between morphology and motility, and the fertilization rate was 74.6% (n=56). It was concluded in general that techniques to evaluate nuclear condensation values do have a high likelihood to give a diagnosis about the future potential of sperm populations in Junín ram.

RETRACTED ARTICLEThe review provides an analysis of domestic and foreign sources devoted to the study of the sperm chromatin structure. The pathogenetic pathways of the sperm DNA fragmentation formation are described. The relationship between sperm DNA damage in pregnancy, live birth rate and the recurrent pregnancy losses in the assisted reproductive technique are presented. The prognostic determination's value of the sperm DNA fragmentation in male infertility cases is noted.

Male fertility throughout life hinges on the successful production of motile sperm, a developmental process that involves three coordinated transitions: mitosis, meiosis, and spermiogenesis. Germ cells undergo both mitosis and meiosis to generate haploid round spermatids, in which histones bound to the male genome are replaced with small nuclear proteins known as protamines. During this transformation, the chromatin undergoes extensive remodeling to become highly compacted in the sperm head. Despite its central role in spermiogenesis and fertility, we lack a comprehensive understanding of the molecular mechanisms underlying the remodeling process, including which remodelers/chaperones are involved, and whether intermediate chromatin proteins function as discrete steps, or unite simultaneously to drive successful exchange. Furthermore, it remains largely unknown whether more nuanced interactions instructed by protamine post-translational modifications affect chromatin dynamics or gene expression in the early embryo. Here, we bring together past and more recent work to explore these topics and suggest future studies that will elevate our understanding of the molecular basis of the histone-to-protamine exchange and the underlying etiology of idiopathic male infertility. [ABSTRACT FROM AUTHOR]

Epigenetic modifications play a special role in the male infertility aetiology. Published data indicate the link between sperm quality and sperm chromatin protamination. This study aimed to determine the relationship between methylation (5mC) and hydroxymethylation (5hmC) in sperm DNA, with respect to sperm chromatin protamination in three subpopulations of fertile normozoospermic controls and infertile patients with oligo-/oligoasthenozoospermia. For the first time, a sequential staining protocol was applied, which allowed researchers to analyse 5mC/5hmC levels by immunofluorescence staining, with a previously determined chromatin protamination status (aniline blue staining), using the same spermatozoa. TUNEL assay determined the sperm DNA fragmentation level. The 5mC/5hmC levels were diversified with respect to chromatin protamination status in both studied groups of males, with the highest values observed in protaminated spermatozoa. The linkage between chromatin protamination and 5mC/5hmC levels in control males disappeared in patients with deteriorated semen parameters. A relationship between 5mC/5hmC and sperm motility/morphology was identified in the patient group. Measuring the 5mC/5hmC status of sperm DNA according to sperm chromatin integrity provides evidence of correct spermatogenesis, and its disruption may represent a prognostic marker for reproductive failure. [ABSTRACT FROM AUTHOR]

Chromium (VI) is the most dangerous oxidation state among the stable forms of chromium. In this work, we evaluated the effect of exposing Mytilus galloprovincialis for 24 h to 1, 10, and 100 nM chromium (VI) on the properties of Protamine-like (PLs) and their gene levels in the gonads. Specifically, we analyzed, by AU-PAGE and SDS-PAGE, PLs extracted from unexposed and exposed mussels. In addition, via EMSA, we evaluated the ability of PLs to bind DNA and also verified their potential to protect DNA from oxidative damage. Finally, we assessed possible alterations in gonadal expression of mt10, hsp70, and genes encoding for PLs-II/PL-IV and PL-III. We found that for all experimental approaches the most relevant alterations occurred after exposure to 1 nM Cr(VI). In particular, a comigration of PL-II with PL-III was observed by SDS-PAGE; and a reduced ability of PLs to bind and protect DNA from oxidative damage was recorded. This dose of chromium (VI) exposure was also the one that produced the greatest alterations in the expression of both mt10 and PL-II/PL-IV encoding genes. All of these changes suggest that this dose of chromium (VI) exposure could affect the reproductive health of Mytilus galloprovincialis. [ABSTRACT FROM AUTHOR]

Copyright of Kafkas Universitesi Veteriner Fakultesi Dergisi is the property of University of Kafkas, Faculty of Veterinary Medicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Background Spermatogenesis is the process by which germ cells develop into spermatozoa in the testis. Sperm protamines are small, arginine-rich nuclear proteins which replace somatic histones during spermatogenesis, allowing a hypercondensed DNA state that leads to a smaller nucleus and facilitating sperm head formation. In eutherian mammals, the protamine-DNA complex is achieved through a combination of intra- and intermolecular cysteine cross-linking and possibly histidine-cysteine zinc ion binding. Most metatherian sperm protamines lack cysteine but perform the same function. This lack of dicysteine cross-linking has made the mechanism behind metatherian protamines folding unclear. Results Protamine sequences from UniProt’s databases were pulled down and sorted into homologous groups. Multiple sequence alignments were then generated and a gap weighted relative entropy score calculated for each position. For the eutherian alignments, the cysteine containing positions were the most highly conserved. For the metatherian alignment, the tyrosine containing positions were the most highly conserved and corresponded to the cysteine positions in the eutherian alignment. Conclusions High conservation indicates likely functionally/structurally important residues at these positions in the metatherian protamines and the correspondence with cysteine positions within the eutherian alignment implies a similarity in function. One possible explanation is that the metatherian protamine structure relies upon dityrosine cross-linking between these highly conserved tyrosines. Also, the human protamine P1 sequence has a tyrosine substitution in a position expecting eutherian dicysteine cross-linking. Similarly, some members of the metatherian Planigales genus contain cysteine substitutions in positions expecting plausible metatherian dityrosine cross-linking. Rare cysteine-tyrosine cross-linking could explain both observations.

Male fertility throughout life hinges on the successful production of motile sperm, a developmental process that involves three coordinated transitions: mitosis, meiosis, and spermiogenesis. Germ cells undergo both mitosis and meiosis to generate haploid round spermatids, in which histones bound to the male genome are replaced with small nuclear proteins known as protamines. During this transformation, the chromatin undergoes extensive remodeling to become highly compacted in the sperm head. Despite its central role in spermiogenesis and fertility, we lack a comprehensive understanding of the molecular mechanisms underlying the remodeling process, including which remodelers/chaperones are involved, and whether intermediate chromatin proteins function as discrete steps, or unite simultaneously to drive successful exchange. Furthermore, it remains largely unknown whether more nuanced interactions instructed by protamine post-translational modifications affect chromatin dynamics or gene expression in the early embryo. Here, we bring together past and more recent work to explore these topics and suggest future studies that will elevate our understanding of the molecular basis of the histone-to-protamine exchange and the underlying etiology of idiopathic male infertility.

Background: Environmental stressors like radiation are a cause for male infertility. The quantum of UV-A in solar ultraviolet (UV) radiation surpasses the amount of UVB by 10 to 100 times, penetrates deeper than UV-B into tissue and mediates possible molecular damage, which warrants evaluation of the impact of UV-A on human spermatozoa. Aim: To determine the differences in effects and responses of UV-A exposure on sperm cells from fertile volunteers and infertile men. Materials and Methods: The study subjects were divided into Group I (Control) and Group II (Infertile). The impact of UV-A on sperm viability, nuclear integrity, chromatin maturity and DNA fragmentation (pre-and post-exposure) was evaluated using standard techniques. Antioxidant enzyme activity assays viz. Lipid Peroxidation (LPO), Superoxide Dismutase (SOD) and Catalase were also analysed. Statistical significance was considered at p < 0.05. Results: This study revealed significant decline in%viability in Group II after UV-A exposure when compared with pre-exposed Group I. The percent sperm with disrupted nuclear toroidal assembly significantly increased in infertile group after exposure, leaving the DNA vulnerable to damage. A significant elevation was observed in the percent immature forms correlated with the significant reduction in% halo formation (impaired nuclear chromatin decondensation) in both post-treated groups as compared to pre-treated groups, suggesting that disruption of sperm toroid triggers DNA damage. UV-A exposure resulted in increased LPO with decrease in antioxidant enzymes in these groups. The percent decline in viability, chromatin decondensation (halo) and antioxidant enzymes were higher in Group II than in Group I. In addition, the percent increase in disrupted toroids and immature chromatin in Group II suggested that the deleterious impact of UV-A on spermatozoa from the infertile group is significantly greater than that of fertile individuals. Conclusion: This study thus revealed that short term UV-A exposure adversely impacts spermatozoa of infertile males to a greater extent than that of the fertile group. Thus, specific awareness and precautions are imperative for occupationally exposed individuals.

Copyright of Revista Cientifica de la Facultade de Veterinaria is the property of Universidad del Zulia, Facultad de Ciencias Veterinarias and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Assisted reproductive technologies involving the use of spermatozoa and eggs for in vitro fertilization (IVF) have come as the solution for many infertile couples to become parents. However, in some cases, the use of ejaculated spermatozoa delivers poor IVF performance. Some studies have suggested the use of testicular spermatozoa in severe male infertility cases, but no guidelines regarding their utilization are currently available. In the present study, we found the mRNA protamine 1/protamine 2 (P1/P2) ratio to be a valuable biomarker of poor sperm function that could be used as a diagnostic key for the identification of cases that would benefit from the use of testicular spermatozoa. A total of 23 couples undergoing egg donation cycles with at least one previous cycle failure were studied. All couples underwent two consecutive intracytoplasmic sperm injection (ICSI) cycles with either ejaculated or testicular spermatozoa (TESA). The sperm mRNA P1/P2 ratio, fertilization rate, blastocyst rate, and pregnancy and live birth rate were compared. Results showed improved ICSI and clinical outcomes in cycles with testicular spermatozoa in men with altered mRNA P1/P2 ratios. TESA cycles presented significantly higher rates of fertilization (mean ± standard deviation: 76.1% ± 15.1% vs 65.5% ± 18.8%), blastocyst formation (55.0% ± 20.3% vs 30.8% ± 23.8%), and good morphological quality blastocyst (28.9% ± 22.9% vs 13.5% ± 17.9%) and also improvements on pregnancy (60.9% vs 0%) and healthy birth rates (56.5% vs 0%) than EJACULATE cycles. The results described here suggest that in patients with previous IVF/ICSI failures and aberrant mRNA protamine ratios, the use of testicular spermatozoa may be a good alternative to improve clinical outcomes.

The sperm nucleus is prone to sustain DNA damage before and after ejaculation. Distribution of the damage is not homogeneous, and the factors determining differential sensitivity among nuclear regions have not yet been characterized. Human sperm chromatin contains three structural domains, two of which are considered the most susceptible to DNA damage: the histone bound domain, harboring developmental related genes, and the domain associated with nuclear matrix proteins. Using a quantitative polymerase chain reaction (qPCR) approach, we analyzed the number of lesions in genes homeobox A3 (HOXA3), homeobox B5 (HOXB 5), sex-determining region Y (SRY)-box 2 (SOX2), β-GLOBIN, rDNA 18S, and rDNA 28S in human sperm after ultraviolet irradiation (400 μW cm−2, 10 min), H2O2treatment (250 mmol l−1, 20 min), and cryopreservation, which showed differential susceptibility to genetic damage. Differential vulnerability is dependent on the genotoxic agent and independent of the sperm nuclear proteins to which the chromatin is bound and of accessibility to the transcription machinery. Immunodetection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that the highest level of oxidation was observed after H2O2treatment. The distribution of oxidative lesions also differed depending on the genotoxic agent. 8-OHdG did not colocalize either with histone 3 (H3) or with type IIα + β topoisomerase (TOPO IIα + β) after H2O2treatment but matched perfectly with peroxiredoxin 6 (PRDX6), which is involved in H2O2metabolism. Our study reveals that the characteristics of the sperm head domains are responsible for access of the genotoxicants and cause differential degree of damage to nuclear areas, whereas chromatin packaging has a very limited relevance. The histone-enriched genes analyzed cannot be used as biomarkers of oxidative DNA damage.

Sperm chromatin not only carries genetic information such as paternal DNA, but also carries structural proteins, epigenetic information, and higher-order chromatin structures (such as matrix attachment regions and telomeres), etc. These information play an important role in embryonic development. This article mainly reviews the effects of these different information carried by sperm chromatin on sperm function and embryonic development and the research progress of related detection methods, in order to provide a theoretical basis and scientific diagnosis and treatment strategies for the etiology screening of clinical infertility, embryo arrest and recurrent miscarriage, so as to improve the pregnancy outcomes of natural conception and assisted reproduction. Keywords: sperm chromatin; epigenetics; sperm DNA damage; sperm function; higher-order chromatin structures.

Background: Prescribing antidepressant drugs is becoming common. These drugs are known to affect sexual functions. Objective: The study is aimed to assess the effects of amitriptyline and venlafaxine on sperm parameters and evaluate Malondialdehyde (MDA) and 1, 1-Diphenyl-2-picryl-hydrazyl values in BALB/ mice spermatozoa. Materials and Methods: Forty adult male BALB/c mice were separated into five groups. Group Ι (control) received distilled water; group ΙΙ amitriptyline (4 mg/kg); group ΙΙΙ amitriptyline (4 mg/kg) +vitamin C (10 mg/kg); group ΙV venlafaxine (2 mg/kg); and group V received vitamin C (10 mg/kg) + venlafaxine (2 mg/kg). All drugs were administered by oral gavage for 35 days. After excision of caudal epididymis, it was located in 1 mL Ham's F10 medium at 37oC for 15 min and then analysis of sperm parameters was performed. To examine lipid peroxidation and total antioxidant capacity, the MDA and 1, 1-Diphenyl-2-picryl-hydrazyl were measured, respectively. Results: The mean sperm parameters in the group treated with amitriptyline were significantly lower than in the other groups. MDA tests showed a significant difference between amitriptyline and control groups (p=0.007). Conclusion: The results of this study showed that amitriptyline consumption can weaken sperm parameters, which can be attributed to the increased production of ROS and toxicity resulting from amitriptyline consumption. Moreover, venlafaxine improved sperm parameters in mice and the lipid peroxidation in this group did not change compared to the control group.

Background: Formalin 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. Objective: This 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. Conclusion: LC can reduce the negative effects of formalin on sperm parameters, chromatin stability, and percentage of apoptosis in an animal model. [ABSTRACT FROM AUTHOR]

Centrifugation of boar semen through one layer of 40% colloid (Porcicoll) was previously shown to separate spermatozoa from bacteria without having a detrimental effect on sperm quality. However, some spermatozoa were lost. The purpose of the present study was to determine whether 20% or 30% Porcicoll could be used to recover most of the spermatozoa without impacting on sperm quality. Insemination doses (n = 10) from a commercial boar station were sent to the laboratory at the Swedish University of Agricultural Sciences and processed by Single Layer Centrifugation with 20% and 30% Porcicoll approximately 7 hr after semen collection. The resulting sperm samples and controls were evaluated for sperm quality immediately and again after storage at 16–18°C for 4 and 7 days. Sperm recovery was 94 ± 18% and 87 ± 15% for 20% and 30% Porcicoll, respectively (p >.05). Sperm mitochondrial membrane potential and chromatin integrity were unaffected (p >.05). The proportion of live spermatozoa producing superoxide (9 ± 8%, 7 ± 6% and 3 ± 1%; p <.05), and the proportion of spermatozoa with high stainability DNA (0.68 ± 19%, 0.61 ± 0.22% and 0.96 ± 0.23%; p <.05‐ <0.01), were marginally increased whereas membrane integrity, although high, was lower in the centrifuged samples than in the controls (82 ± 8%, 83 ± 5% versus 92 ± 4%; p <.05). In conclusion, centrifugation through 20% or 30% Porcicoll enables most spermatozoa to be recovered, without having a major effect on sperm quality. These results are encouraging for further studies involving microbiological investigation of the processed samples, and scaling‐up to process larger volumes of boar ejaculates. [ABSTRACT FROM AUTHOR]

The sperm nucleus is prone to sustain DNA damage before and after ejaculation. Distribution of the damage is not homogeneous, and the factors determining differential sensitivity among nuclear regions have not yet been characterized. Human sperm chromatin contains three structural domains, two of which are considered the most susceptible to DNA damage: the histone bound domain, harboring developmental related genes, and the domain associated with nuclear matrix proteins. Using a quantitative polymerase chain reaction (qPCR) approach, we analyzed the number of lesions in genes homeobox A3 (HOXA3), homeobox B5 (HOXB5), sex-determining region Y (SRY)-box 2 (SOX2), β-GLOBIN, rDNA 18S, and rDNA 28S in human sperm after ultraviolet irradiation (400 µW cm-2, 10 min), H2O2 treatment (250 mmol l-1, 20 min), and cryopreservation, which showed differential susceptibility to genetic damage. Differential vulnerability is dependent on the genotoxic agent and independent of the sperm nuclear proteins to which the chromatin is bound and of accessibility to the transcription machinery. Immunodetection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that the highest level of oxidation was observed after H2O2 treatment. The distribution of oxidative lesions also differed depending on the genotoxic agent. 8-OHdG did not colocalize either with histone 3 (H3) or with type IIα + β topoisomerase (TOPO IIα + β) after H2O2 treatment but matched perfectly with peroxiredoxin 6 (PRDX6), which is involved in H2O2 metabolism. Our study reveals that the characteristics of the sperm head domains are responsible for access of the genotoxicants and cause differential degree of damage to nuclear areas, whereas chromatin packaging has a very limited relevance. The histone-enriched genes analyzed cannot be used as biomarkers of oxidative DNA damage. [ABSTRACT FROM AUTHOR]

Background: Spermatogenesis is the process by which germ cells develop into spermatozoa in the testis. Sperm protamines are small, arginine-rich nuclear proteins which replace somatic histones during spermatogenesis, allowing a hypercondensed DNA state that leads to a smaller nucleus and facilitating sperm head formation. In eutherian mammals, the protamine-DNA complex is achieved through a combination of intra- and intermolecular cysteine cross-linking and possibly histidine-cysteine zinc ion binding. Most metatherian sperm protamines lack cysteine but perform the same function. This lack of dicysteine cross-linking has made the mechanism behind metatherian protamines folding unclear. Results: Protamine sequences from UniProt's databases were pulled down and sorted into homologous groups. Multiple sequence alignments were then generated and a gap weighted relative entropy score calculated for each position. For the eutherian alignments, the cysteine containing positions were the most highly conserved. For the metatherian alignment, the tyrosine containing positions were the most highly conserved and corresponded to the cysteine positions in the eutherian alignment. Conclusions: High conservation indicates likely functionally/structurally important residues at these positions in the metatherian protamines and the correspondence with cysteine positions within the eutherian alignment implies a similarity in function. One possible explanation is that the metatherian protamine structure relies upon dityrosine cross-linking between these highly conserved tyrosines. Also, the human protamine P1 sequence has a tyrosine substitution in a position expecting eutherian dicysteine cross-linking. Similarly, some members of the metatherian Planigales genus contain cysteine substitutions in positions expecting plausible metatherian dityrosine cross-linking. Rare cysteine-tyrosine cross-linking could explain both observations. [ABSTRACT FROM AUTHOR]

F2-isoprostanes (F2-IsoPs), stereoisomers of prostaglandin F2α generated by the free radical-induced oxidation of arachidonic acid, have been associated with different male infertility conditions. This study aimed to evaluate the role of seminal isoprostane levels and sperm characteristics in the reproductive outcome and embryo quality of 49 infertile couples. Semen analysis was performed following WHO guidelines. Sperm chromatin maturity was detected using an aniline blue (AB) assay, and DNA integrity was assessed using the acridine orange (AO) test. Seminal F2-IsoP levels were quantified by gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI–MS/MS) analysis. Correlations among variables and their impact on in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) outcome were investigated. F2-IsoP levels are positively correlated with double-stranded DNA sperm (p < 0.001) and negatively correlated with mature sperm chromatin (p < 0.001). Patients with positive outcomes had an increased percentage of sperm with double-stranded DNA, as did patients producing high-quality embryo, who showed higher F2-IsoP levels compared to those detected in the low-quality embryo group. An intriguing relationship between a mild increase in F2-IsoP levels, DNA integrity, and embryo quality seems to indicate that the non-enzymatic oxidation of arachidonic acid can be also a marker of metabolic activity in human semen.

Summary: Sperm DNA fragmentation is referred to as one of the main causes of male infertility. Failures in the protamination process, apoptosis and action of reactive oxygen species (ROS) are considered the most important causes of DNA fragmentation. Action of ROS or changes in sperm protamination would increase the susceptibility of sperm DNA to fragmentation. Routine semen analysis is unable to estimate sperm chromatin damage. Sperm DNA integrity influences sperm functional capability, therefore tests that measure sperm DNA fragmentation are important to assess fertility disorders. Actually, there is a considerable number of methods for assessing sperm DNA fragmentation and chromatin integrity, sperm chromatin stability assay (SCSA modified), sperm chromatin dispersion (SCD), comet assay, transferase dUTP nick end labelling (TUNEL); and protamine evaluation in sperm chromatin assay, such as toluidine blue, CMA3, protamine expression and evaluation of cysteine radicals. This review aims to describe the main causes of sperm DNA fragmentation and the tests commonly used to evaluate sperm DNA fragmentation. [ABSTRACT FROM AUTHOR]

Background: Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa. Objectives: The aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis. Materials and methods: For this purpose, absolute q‐RT‐PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry. Results: We evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results. Conclusion: Our work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility. [ABSTRACT FROM AUTHOR]

Summary: Direct swim-up procedure is widely used to separate the motile competent spermatozoa from the antioxidant-rich semen. Subsequently, spermatozoa become more vulnerable to reactive oxygen species (ROS) due to their cytological characteristics. The effect of vitamin C, a highly concentrated antioxidant in the semen, on direct swim-up-enriched sperm population is not fully investigated. Therefore, the aim of the present study was to assess the effect of vitamin C on sperm functional properties during direct swim-up procedure. Semen samples were collected from 22 participants. Each semen sample was divided into several aliquots. The first portion was overlaid with sperm medium without ascorbic acid (0 µM AA). The second and third fractions were overlaid with sperm medium supplemented with 300 µM and 600 µM AA; respectively. After 1 h of incubation, basic sperm parameters, intracellular ROS levels, acrosome reaction, chromatin integrity, and glucose uptake were assessed. Swim-up without AA significantly increased the percentage of ROS(+) spermatozoa compared with the raw semen (P <0.01). Interestingly, swim-up with 300 µM AA did not increase the percentage of ROS(+) sperm compared with the raw semen. In parallel, the percentage of sperm with altered chromatin integrity was significantly lower in the 300 µM AA group compared with that in the raw semen (P <0.05). These findings suggest that supplementation of vitamin C to sperm medium could be beneficial for direct swim-up-derived spermatozoa. [ABSTRACT FROM AUTHOR]

A large number of heavy metals resulted toxic to the reproductive system, but invertebrate infertility has been poorly explored, and above all, there are limited molecular, cellular and toxicological studies. In the present work, we exposed Mytilus galloprovincialis to three individual metal chlorides (CuCl 2 15 μM, CdCl 2 1.5 μM, NiCl 2 15 μM) and their mixture for 24 h, to evaluate the effects on the protamine-like proteins (PLs), sperm DNA and on their interaction in the formation of sperm chromatin. Under all exposure conditions, but particularly after exposure to the metals mix, relevant changes in the electrophoretic pattern, by AU-PAGE and SDS-PAGE, and in fluorescence spectroscopy measurements of PLs were shown. In addition, alterations in DNA binding of these proteins were observed by Electrophoretic Mobility Shift Assay (EMSA) and through their release from sperm nuclei. Moreover, there was evidence of increased accessibility of micrococcal nuclease to sperm chromatin, which was also confirmed by toluidine blue staining. Furthermore, morphological analyses indicated severe gonadal impairments which was also corroborated by increased PARP expression, by Western blotting, and sperm DNA fragmentation, by comet assay. Finally, we investigated the expression of stress genes, gst , hsp70 and mt10 , in gonadal tissue. The latter investigations also showed that exposure to this metals mix was more harmful than exposure to the individual metals tested. The present results suggest that these metals and in particular their mixture could have a negative impact on the reproductive fitness of M. galloprovincialis. Based on these evidences, we propose a molecular mechanism. [Display omitted] • PLs-DNA binding is affected by mussels exposure to specific mixtures of heavy metals • Interaction between these heavy metals enhances DNA damage • Heavy metals and their mixture cause a disconnection of germ cells in gonads • Possible toxicological mechanisms of heavy metals action on M. galloprovincialis • Heavy metals cause a different chromatin condensation in spermatozoa [ABSTRACT FROM AUTHOR]

Objective: To evaluate the effects of low level ultraviolet (UV) radiation on sperm chromatin structure.Methods: The target study was divided into three groups: (i) males with proven fertility (n=40 ) was taken as Group I (control); (ii) Oligoasthenozoospermic (OAT) cases as Group II (n=36); (iii) males with unexplained infertility (MUI) cases (n=42) as Group III. Specific techniques were used to study the impact of UV radiation (Pre and Post UV exposure) on the sperm nuclear DNA viz. Aniline blue staining was for detection of immature chromatin. Chromomycin A3 fluorescence staining was used to determine protamine-DNA dissociation by intense fluorescence of protamine deficient sperm cells and neutral comet assay was for evaluation of DNA fragmentation. Statistical analysis was carried out using Student's t-test (GraphPad Prism Version-6). Level of significance was considered at P