Sperm Chromatin Condensation Research Articles

6 ALTERED CHROMATIN CONFORMATION IN BOVINE SPERMATOZOA PERTURBS DYNAMIC DNA METHYLATION IN THE MALE PRONUCLEUS AFTER FERTILIZATION IN VITRO

Soon after fertilization, mammalian zygotes need proper DNA methylation reprogramming, at which time the epigenetic marks that the oocyte and sperm have acquired during gametogenesis are erased to allow totipotent zygotic development. Aberrant epigenetic marks in the paternal genome are thought to be associated with altered chromatin condensation in spermatozoa of suboptimal quality. We have recently reported that heat stress on bulls during germ cell development, especially at the spermiogenesis stage, altered sperm chromatin condensation. The objective of this study was to investigate dynamic DNA methylation reprogramming in the male pronucleus after fertilization of oocytes with sperm known to have altered chromatin conformation. To evaluate dynamic DNA methylation reprogramming, zygotes collected at 3 different time points [i.e. 12, 18, and 24 h post-insemination (hpi)] were immunocytochemically investigated using an antibody against 5-methylcytosine (5mC). The total fluorescence intensity of the male pronuclei (n = 89, ≥25 in each group) was measured by ImageJ and data were analyzed by ANOVA. The DNA methylation pattern in male pronuclei when oocytes were fertilized with heat-stressed sperm did not change between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. The results of this study indicated that dynamic DNA methylation reprogramming patterns such as DNA demethylation followed by de novo methylation in the male pronucleus soon after fertilization were altered when oocytes were fertilized with heat-stressed sperm. In conclusion, altered sperm chromatin conformation due to heat stress perturbs dynamic DNA methylation reprogramming in the male pronucleus, which may hamper nuclear totipotency and embryo survival.

Putative molecular mechanism underlying sperm chromatin remodelling is regulated by reproductive hormones

BackgroundThe putative regulatory role of the male reproductive hormones in the molecular mechanism underlying chromatin condensation remains poorly understood. In the past decade, we developed two adult male rat models wherein functional deficits of testosterone or FSH, produced after treatments with 20 mg/Kg/d of cyproterone acetate (CPA) per os, for a period of 15 days or 3 mg/Kg/d of fluphenazine decanoate (FD) subcutaneously, for a period of 60 days, respectively, affected the rate of sperm chromatin decondensation in vitro. These rat models have been used in the current study in order to delineate the putative roles of testosterone and FSH in the molecular mechanism underlying remodelling of sperm chromatin.ResultsWe report that deficits of both testosterone and FSH affected the turnover of polyubiquitylated histones and led to their accumulation in the testis. Functional deficits of testosterone reduced expression of MIWI, the 5-methyl cap binding RNA-binding protein (PIWIlike murine homologue of the Drosophila protein PIWI/P-element induced wimpy testis) containing a PAZ/Piwi-Argonaut-Zwille domain and levels of histone deacetylase1 (HDAC1), ubiquitin ligating enzyme (URE-B1/E3), 20S proteasome α1 concomitant with reduced expression of ubiquitin activating enzyme (ube1), conjugating enzyme (ube2d2), chromodomain Y like protein (cdyl), bromodomain testis specific protein (brdt), hdac6 (histone deacetylase6), androgen-dependent homeobox placentae embryonic protein (pem/RhoX5), histones h2b and th3 (testis-specific h3). Functional deficits of FSH reduced the expression of cdyl and brdt genes in the testis, affected turnover of ubiquitylated histones, stalled the physiological DNA repair mechanism and culminated in spermiation of DNA damaged sperm.ConclusionsWe aver that deficits of both testosterone and FSH differentially affected the process of sperm chromatin remodelling through subtle changes in the ‘chromatin condensation transcriptome and proteome’, thereby stalling the replacement of ‘dynamic’ histones with ‘inert’ protamines, and altering the epigenetic state of condensed sperm chromatin. The inappropriately condensed chromatin affected the sperm chromatin cytoarchitecture, evident from subtle ultrastructural changes in the nuclei of immature caput epididymal sperm of CPA- or FD-treated rats, incubated in vitro with dithiothreitol.

Dynamics of Sperm Chromatin Associated with Bull Fertility.

Genomic DNA of bull spermatozoa is tightly packaged mostly around Protamine 1 (PRM1) and small amounts of histones such as histone3 (H3) in a much smaller volume compared to somatic cells. Proper packaging of genome is important both for sperm morphology and physiology as well as for fertilization and embryonic development. Both PRM1 and histone3.3 (H3.3; a variant of H3) are known to play important roles during sperm chromatin condensation. Recently, H3.3 has been shown to be bound to actively transcribed regions of the paternal genome; therefore, its presence becomes important. However, molecular and cellular underpinnings of their functions in bull spermatozoa and how these two proteins regulate fertility are not known. The objective of this study was to determine the dynamics of chromatin structure and the expression patterns of DNA binding proteins PRM1 and H3.3 in spermatozoa from bulls with varying fertility. To examine sperm chromatin condensation, chromatin integrity and protamination were detected using halomax assay and toluidine blue staining in spermatozoa from 20 bulls with distinct fertility, which was equally grouped into low and high fertility. For both methods, three biological and two technical replicates were performed per bull, and 500 spermatozoa were counted per slide by two different technicians using light microscopy. We also performed western blotting to detect expression levels of PRM1 and H3.3 followed by acetic acid-urea gel system and computational biology to determine functional motifs in the two proteins. In addition, spermatozoa from three bulls per group were used to conduct IVF experiments with three replicates. Statistical analysis was performed using SAS. Our results revealed that sperm chromatin integrity 7.01% (±2.24) vs. 3.97% (±0.95); p < 0.0001 and protamination 3.27% (±0.99) vs.1.2% (±0.91); p < 0.0001 were significantly different between low and high fertility groups, respectively. Based on the IVF results, embryo cleavage and development rates to blastocyst stage were lower in low fertility group compared to their high fertility counterparts 57.56% (±10.21) vs. 68.92% (±11.65); p <0.05 and 11.7% (± 3.50) vs. 16.96% (± 6.51); p < 0.05, respectively. We confirmed that slide readings of halomax and toluidine blue experiments were not different between the two technicians (p > 0.54 and > 0.80, respectively). We also found that in vivo fertility scores of the bulls were negatively correlated with sperm chromatin protamination (r= −0.62; p < 0.0001) and integrity (r= −0.69; p < 0.0001). In addition, sperm chromatin integrity among the bulls was negatively associated with embryo cleavage (r= −0.56; p < 0.05) and development rate (r= −0.45; p = 0.0637) in embryos derived using spermatozoa from the bulls with varying fertility. In contrast to PRM1, functional motifs were only found in H3 such as histone H3.2, CAMP phospho site, myristyl and Ck2 phospho. We also showed that there is a ratio between the protein expression levels of PRM1 and H3.3 among the bulls. In conclusion, our data showed that inadequate sperm chromatin protamination and integrity were associated with inefficient sperm chromatin condensation leading to improper fertilization and beyond, which can be estimated using bull fertility scores prior to use them for AI. Our results provide a comprehensive output in sperm chromatin dynamics that impacts sperm viability, fertilization and early embryonic development.

Establishment of Normal Sperm Nuclear Architecture Requires Poly(ADP-Ribose) Metabolism During Spermatogenesis.

Spermatozoa differ dramatically from somatic cells with respect to their chromatin composition and nuclear architecture. Sperm DNA is predominantly organized in a highly condensed form that is achieved through complexation of DNA with protamines. Only a minor portion of the sperm genome retains the nucleosomal organization present in somatic cells. Mammalian sperm are also characterized by their unique and highly ordered nuclear architecture. The pericentromeric heterochromatin regions of individual chromosomes colocalize to form a centrally positioned heterochromatin block (“chromocenter”), and telomeric regions of chromosomes are positioned pairwise at the nuclear periphery. The resulting highly ordered structure is characterized by individual chromosomes occupying defined regions that stretch from the chromocenter to the nuclear envelope. The mechanisms that govern establishment and maintenance of the specific sperm nuclear architecture, including the formation of the characteristic chromocenter during spermiogenesis are not yet well understood. We previously reported that the exchange of histones to protamines, which necessitates concurrent DNA relaxation, involves the coordinated activities of topoisomerase 2 beta and poly(ADP-ribose) polymerase (PARP). Inhibition of PARP activity using a genetically altered mouse model as well as a pharmacological PARP inhibitor during spermiogenesis reduced the degree of sperm chromatin condensation and increased levels of residual histones. Here, we investigated the impact of PARP inhibition on sperm nuclear architecture using fluorescence in situ hybridization (FISH) of pericentric heterochromatin regions and volumetric measurements employing confocal imaging. Reduced PAR metabolism resulted in significantly smaller volumes occupied by the pancentromeric heterochromatin constituting the chromocenter and the fragmentation of the chromocenter into several individual heterochromatic clusters was detected. We conclude that PAR metabolism during spermiogenesis not only promotes formation of proper sperm nucleoprotein composition, but also influences establishment of structural aspects of sperm nuclear architecture. The specialized nuclear structure of spermatozoa may be functionally relevant for the reorganization of the paternal genome after fertilization, and early embryonic gene expression. Because levels of cellular PAR formation depend directly on environmental factors (i.e. the exposure to genotoxic stressors), we propose that the PAR pathway may be capable of altering sperm epigenetic information in response to environmental factors. Research supported by NIH/NICHD HD48837 to RGM.

Sperm selection based on motility in polyvinylpyrrolidone is associated with successful pregnancy and embryo development

The aim of this study was to investigate whether spermatozoon motility in polyvinylpyrrolidone (PVP) is associated with better embryo development and pregnancy rates in ICSI cycles. A total of 123 primary ICSI treatment cycles were included in this study. Semen samples were tested for motility before ICSI procedure in PVP. Within 3min, the presence or absence of motility was recorded. Sperm functions were examined by the aniline blue (AB) chromatin condensation test and the hypoosmotic swelling test, and the chromatin stability was evaluated by inducing its decondensation with sodium dodecyl sulphate and ethylenediaminetetraacetic acid (EDTA). Fertilisation and embryo scoring were evaluated. Fifty (64%) of 78 women conceived in the PVP (+) group; and 12 (26%) of 45 women conceived in the PVP (-) group; the pregnancy rate was significantly higher in the PVP (+) group (P=0.003). Semen parameters were observed to be similar in both groups. The mean number of total embryos obtained in ICSI procedure and transferred grade 1 embryos were significantly higher in PVP (+) group (P=0.01 and P=0.003 respectively). The presence of sperm motility in PVP is associated with increased pregnancy rate, higher percentage of good quality embryos, sperm chromatin condensation and decondensation.

Effects of Varicocelectomy on Sperm DNA Fragmentation, Mitochondrial Function, Chromatin Condensation, and Apoptosis

The aim of this study was to evaluate conventional semen parameters (density, morphology, and progressive motility) and the flow-cytometric parameters of DNA fragmentation, mitochondrial membrane potential, phosphatidylserine externalization, and chromatin compactness in patients with varicocele before and after varicocelectomy. Thirty men (26.5 ± 3.2 years old, range 20-32 years) with oligoasthenoteratozoospermia and grade 3 left varicocele were selected (without other causes of male infertility). Each of them underwent sperm analysis and flow cytometric evaluation before and 4 months after subinguinal microsurgical varicocelectomy (SMV). After varicocelectomy, men had significantly higher sperm density, progressive motility, and normal forms compared with baseline. They also had a significantly lower percentage of spermatozoa with low mitochondrial membrane potential. After SMV, they showed a significantly lower percentage of spermatozoa with phosphatidylserine externalization, an early sign of apoptosis. Significantly decreased percentages of spermatozoa with abnormal chromatin compactness and spermatozoa with DNA fragmentation were found after SMV compared with baseline. Subinguinal microsurgical varicocelectomy improves sperm function in oligoasthenoteratozoospermia secondary to grade 3 left varicocele. Improvements are seen in conventional parameters and biofunctional parameters not routinely evaluated.

Paternal DNA Damage Resulting From Various Sperm Treatments Persists After Fertilization and Is Similar Before and After DNA Replication

In spite of its highly condensed state, sperm DNA is vulnerable to damage that can originate from oxidative stress, the activity of sperm-specific nucleases, or both. After fertilization, in the oocyte, paternal chromatin undergoes dramatic changes, and during this extensive remodeling, it can be both repaired and degraded, and these processes can be linked to DNA synthesis. Here, we analyzed sperm response to damage-inducing treatments both before and after fertilization and before or after zygotic DNA replication. Epididymal mouse spermatozoa were either frozen without cryoprotection (FT) or treated with detergent Triton X-100 coupled with dithiothreitol (TX+DTT) to induce DNA damage. Fresh, untreated sperm served as control. Immediately after preparation, spermatozoa from 3 groups were taken for comet assay, or for intracytoplasmic sperm injection into prometaphase I oocytes to visualize prematurely condensed single-chromatid chromosomes, or into mature metaphase II oocytes to visualize chromosomes after DNA replication. Comet assay revealed increased DNA fragmentation in treated sperm when compared with control, with FT sperm more severely affected. Chromosome analysis demonstrated paternal DNA damage in oocytes injected with treated, but not with fresh, sperm, with FT and TX+DTT groups now yielding similar damage. There were no differences in the incidence of abnormal paternal karyoplates before and after DNA synthesis in all examined groups. This study provides evidence that subjecting sperm to DNA damage-inducing treatments results in degradation of highly condensed sperm chromatin when it is still packed within the sperm head, and that this DNA damage persists after fertilization. The difference in DNA damage in sperm subjected to 2 treatments was ameliorated in the fertilized oocytes, suggesting that some chromatin repair might have occurred. This process, however, was independent of DNA synthesis and took place during oocyte maturation.

Negative Effect of Increased Body Weight on Sperm Conventional and Nonconventional Flow Cytometric Sperm Parameters

Although with some discrepancy, obese men have been reported to have the worst conventional sperm parameters, but little is known about the effects of body weight on sperm mitochondrial function, chromatin condensation and apoptosis. This study was undertaken to evaluate conventional and nonconventional sperm parameters in nonsmoking overweight or obese men without any other cause known to alter sperm parameters. Fifty overweight, 50 obese, and 50 normal-weight healthy nonsmoking men were carefully selected. Each man underwent up to 2 sperm analyses and evaluation of mitochondrial membrane potential (MMP), phosphatidylserine (PS) externalization, chromatin compactness, and DNA fragmentation by flow cytometry. Overweight and obese men had significantly lower sperm progressive motility and normal forms than controls. They also had a significantly higher percentage of spermatozoa with low MMP. Obese, but not overweight, men showed a significantly higher percentage of spermatozoa with PS externalization, an early sign of apoptosis, and a lower percentage of viable spermatozoa. A significant increased percentage of spermatozoa with abnormal chromatin compactness was found in both overweight and obese men, whereas only obese men had a significantly higher number of spermatozoa with DNA fragmentation compared with controls. Healthy nonsmoking overweight and obese men have worse conventional and nonconventional sperm parameters than normal-weight controls. The important role played by these parameters in a couple's fertility suggests a program of body weight loss among the therapeutic repertoire for male infertility.

119 HETEROLOGOUS BOVINE IN VITRO FERTILIZATION USING CRYOPRESERVED BOTTLENOSE DOLPHIN SPERMATOZOA

Assisted reproductive technologies are of great importance for increasing genetic diversity in captive animals without displacing them. The development and improvement of these techniques require accurate methods to assess sperm function. The ability of the sperm to bind the zona pellucida and the formation of a male pronucleus have been shown to have a high predictive value for fertilization outcome. The use of zona-intact bovine in vitro–matured oocytes in heterologous fertilization with dolphin spermatozoa could provide valuable information on its fertilizing ability. The aim of the present study was to evaluate male pronuclear formation in zona-intact bovine oocytes after coincubation with frozen-thawed bottlenose dolphin spermatozoa. A total of 1546 immature cumulus oocytes complexes (COC) were obtained from bovine ovaries collected at slaughter. The COC were matured for 24 h in TCM-199 supplemented with 10 ng mL–1 of epidermal growth factor and 10% FCS. Matured COC were inseminated with frozen-thawed Bovi-pure (Nidacon International, Mölndal, Sweden) separated bovine (control) or dolphin spermatozoa. At 18, 20, 22, 24, 26 and 28 h post-insemination (hpi), half of the presumptive zygotes from each group were fixed and stained with Hoechst 33342 to examine sperm penetration, polyspermy and pronuclear formation and the remainder were cultured in synthetic oviduct fluid supplemented with 5% FCS for evaluating fertilization rates by cleavage on Days 2 and 4 (Day 0 = day of IVF). As expected, in the control a higher percentage of 2 pronuclear formation was observed at 18 hpi (74.5%), with a decrease at 20 and 22 hpi (57.4 and 43.2%, respectively) and was significantly lower (P ≤ 0.001) at 24 hpi (13.3%), reaching the lowest values at 26 and 28 hpi. However, in the heterologous group significantly less oocytes with both pronuclear formed (P ≤ 0.001) were observed at 18, 20 and 22 hpi (1.2, 3.4 and 3.0%, respectively) compared with 24, 26 and 28 hpi (22.5, 11.4 and 8.9%, respectively). No polyspermy was detected in oocytes coincubated with dolphin spermatozoa. Moreover, the cleavage rate at Day 2 and 4 in heterologous fertilization was 13.0 and 34.8%, respectively, whereas for the control it was 90.0%. In conclusion, these results indicate that dolphin spermatozoa can penetrate bovine oocytes and induce the block to polyspermy and the differences found regarding pronuclear formation times between the 2 species could be due to distinct sperm chromatin organisation or condensation. In conclusion, our preliminary results show that heterologous fertilization using bovine oocytes is useful for characterising the viability of dolphin thawed spermatozoa, which also could be helpful in performing a more complete sperm evaluation. Further studies are necessary to provide more consistent evidence of the efficiency of this test. The authors thank the staff at Zoo Aquarium Madrid for their dedicated work toward dolphin semen collection.

Accurate sperm morphology assessment predicts sperm function

Sperm morphology has been associated with in vitro as well as in vivo fertilisation. The study aimed to evaluate the possible relation between the percentage of spermatozoa with normal morphology and the following sperm functional assays: (i) zona-induced acrosome reaction (ZIAR); (ii) DNA integrity; (iii) chromatin condensation; (iv) sperm apoptosis; and (v) fertilisation rates. Regression analysis was employed to calculate the association between morphology and different functional tests. Normal sperm morphology correlated significantly with the percentages of live acrosome-reacted spermatozoa in the ZIAR (r = 0.518; P < 0.0001; n = 92), DNA integrity (r = -0.515; P = 0.0018; n = 34), CMA(3) -positive spermatozoa (r = -0.745; P < 0.0001; n = 92), sperm apoptosis (r = -0.395; P = 0.0206; n = 34) and necrosis (r = -0.545; P = 0.0009; n = 34). Negative correlations existed between for the acrosome reaction, and DNA integrity, while negative associations were recorded with the percentages of CMA(3) -positive spermatozoa, apoptotic and necrotic spermatozoa. Sperm morphology is related to sperm dysfunction such as poor chromatin condensation, acrosome reaction and DNA integrity. Negative and significant correlations existed between normal sperm morphology and chromatin condensation, the percentage of spermatozoa with abnormal DNA and spermatozoa with apoptotic activity. The authors do not regard sperm morphology as the only test for the diagnosis of male fertility, but sperm morphology can serve as a valuable indicator of underlying dysfunction.

Characteristics of selected seminal plasma proteins and their application in the improvement of the reproductive processes in mammals

Understanding the biochemical processes associated with ovum fertilization and knowledge about the structure and function of individual substances participating in these processes is crucial for the development of biotechnological methods to improve reproduction of animals and humans. Among many components of seminal plasma, proteins and peptides play a specific role in regulation of the fertilization process, particularly through their ability to bind various types of ligands such as polysaccharides, lipids and ions. Heparin-binding proteins regulate capacitation and acrosome reaction processes. Affinity of plasma proteins to mannans of the fallopian tube epithelium facilitates formation of spermatozoa reservoirs in the female reproductive tract. Ability to bind phosphorylcholine is one of the conditions for the coating of the seminal plasma proteins on the sperm membrane and also determines the formation of oligomeric forms of certain proteins. Zinc binding by seminal plasma proteins regulates sperm chromatin condensation state. It also affects motility of these cells and acrosome reaction. The interspecies analysis indicates significant structural and functional similarities, especially for the proteins with low molecular weight. Fertility associated proteins (FAPs) have been determined in the bull, stallion, boar, ram and dog. The contents of these proteins correlate with the indicators of the fertilizing abilities of sperm. In humans, several seminal plasma proteins were found which serve as diagnostic markers of spermatogenesis, seminiferous epithelium state, and azoospermia. To determine the semen ability for preservation, measurement of some seminal plasma protein content may also be used. Addition of specific plasma proteins to a spermatozoa solution undergoing the process of preservation may be used to retain the features of the cells responsible for efficient fertilization.

Effects of testicular sperm chromatin condensation assay using aniline blue-eosin staining in IVF-ET cycle

Effects of testicular sperm chromatin condensation assay using aniline blue-eosin staining in IVF-ET cycle

PCDC2 (Y15) expression during oocyte maturation- implications in fertilization failure after ICSI

To evaluate the expression of pCdc2 during oocyte maturation and fertilization failures after ICSI. Determine the oocyte cytoplasmic competence (pCdc2 levels) with premature sperm chromatin condensation (PCC) incidence.

Assessment of two thawing processes of cryopreserved human sperm in pellets

In this study, we evaluated the effects of the thawing methodology on sperm function after cryopreservation in pellets. We compared the use of two thawing procedures: method (1) maintaining pellet for 10min in air at room temperature, then another 10-min period in air at 37°C followed by dilution in a thawing medium; and method (2) immersing the pellets directly in thawing medium at 37°C for 20min. This procedure leads to a higher rate of temperature increase and a dilution of the glycerol present in the freezing medium. We analyzed the effect of the thawing procedure on sperm motility, viability, membrane lipid packing disorder, acrosome status, reactive oxygen species (ROS) level and sperm chromatin condensation. This study revealed a positive effect of the M2 thawing methodology on sperm parameters. The percentage of spermatozoa with fast-linear movement is increased (M1: 17.26% vs. M2: 28.05%, p<0.01), with higher viability (M1: 37.81% vs. M2: 40.15%, p<0.01) and less acrosome damage (M1: 40.44% vs. M2: 35.45%, p=0.02). We also detected an increase in the percentage of viable spermatozoa with low membrane lipid disorder (M1: 31.36% vs. M2: 33.17%, p=0.03) and a reduction in chromatin condensation (44.62 vs. 46.62 arbitrary units, p=0.02). Further studies will be necessary to evaluate the possible clinical applications.

Inverse correlation between chromatin condensation and sperm head size in a case of enlarged sperm heads

Among sperm morphology abnormalities, macrocephalic and large-headed spermatozoa are commonly associated with a low chance of pregnancy, mainly in relation to meiotic abnormalities during spermatogenesis. Here is reported the case of a patient with 98% of spermatozoa showing abnormal morphology, many having enlarged heads (47%). Sperm-head measurement, sperm fluorescent in situ hybridization analysis, sperm chromatin decondensation and molecular biology were performed. Fifty-six percent of the sperm displayed a large head (length >4.7 μm and width >3.2 μm), and the mean sperm-head area was 15.8 ± 3.8 μm 2 (9.7 ± 1.5 μm 2 and 9.3 ± 1.4 μm 2 for two controls). Normal chromosomal content was found in 97% of the cells and no aurea kinase C-gene mutation was found. Mean sperm chromatin decondensation rate was 46%, 64% for large-head forms and 10% for other forms. This is, as far as is known, one of the first cases of semen with enlarged-head spermatozoa linked to sperm chromatin condensation dysfunction with no major meiotic dysfunction. The study centre advised the couple to undergo intracytoplasmic sperm injection with the patient’s spermatozoa. Among sperm morphology abnormalities, macrocephalic and large-headed spermatozoa are commonly associated with a low chance of pregnancy, mainly in relation to meiotic abnormalities during spermatogenesis. We report the case of a 51-year-old referred to our centre for a 3-year history of secondary infertility who had 98% of spermatozoa with abnormal morphology and many with enlarged sperm heads (47%). Sperm head measurements, sperm fluorescent in situ hybridization analysis, and sperm chromatin decondensation were performed to propose the best treatment for this couple. In contrast with previous studies indicating a high abnormal chromosome content correlated with the percentage of macrocephalic spermatozoa, 97% of the spermatozoa in this case had a normal content for chromosomes analysed. Aniline blue staining showed a high rate of decondensed spermatozoa (46%), compared with controls (19% and 20%). We showed that the mean sperm head area for decondensed sperm heads was greater ( P < 0.0001) than that for condensed sperm heads, and an increased rate ( P < 0.0001) of decondensed sperm heads was found for macrocephalic spermatozoa (>14 μm 2), compared with normal sperm heads; respectively 64% and 10%. Because during the ICSI procedure the operator select apparently normal sperm form (mainly with normal condensed head), we advised, in this particular case, the couple to undergo ICSI with the patient’s spermatozoa as they had a good prognosis compared with previous data published for macrocephalic spermatozoa.

Assessment of sperm chromatin condensation and ploidy status using flow cytometry correlates to fertilization, embryo quality and pregnancy following in vitro fertilization

Sperm flow cytometry (SFC) was used to evaluate the association of sperm chromatin condensation and ploidy with fertilization, embryo development, pregnancy and abortion rates following IVF. Conventional semen analysis was performed in one hundred fifty men, as well as SFC analysis, after acridine orange and propidium iodide staining, for the evaluation of sperm maturity and ploidy respectively. Conventional IVF was performed in all couples. Couples with low percentages of mature spermatozoa presented with lower fertilization rates (p < 0.005), lower rates of grade A embryos (p < 0.003) and lower pregnancy rates (p < 0.006), compared to couples with high percentages of mature spermatozoa. Couples with low total aneuploidy rates presented with higher fertilization rates (p < 0.007), higher rates of grade A embryos (p < 0.004) and higher pregnancy rates (p < 0.003), compared to couples with high total aneuploidy rates. Sperm chromatin condensation and ploidy constitute critical parameters for the evaluation of semen samples before IVF and for the identification of cases in need of ICSI application.

Evaluation of the effect of cooling and of the addition of collagenase on llama sperm DNA using toluidine blue

The effect cryopreservation has on sperm chromatin condensation has been studied in many species but not in South American camelids. The objectives of this study were to evaluate with toluidine blue (TB) the effects of cooling and of adding collagenase on llama sperm DNA condensation. The optimum incubation time (30 s, 1.5 and 3 min) with a reducing agent (dithiothreitol) was also determined. When comparing cooled samples with the raw ejaculate, a significant increase in sperm showing a high degree of decondensation (TB positive) was observed (P = 0.005). A positive correlation was observed, both in raw and cooled semen, between sperm head morphological abnormalities observed in TB-stained cells and TB-positive sperm (highly decondensed DNA), but not with TB-intermediate spermatozoa (moderately decondensed DNA). No significant differences (P > 0.05) were observed in samples incubated with or without 0.1% collagenase. In cooled semen, but not in raw, a significant increase (P = 0.000) in reacted sperm (TB positive) was observed using 3-min incubation with 1% dithiothreitol (DTT). To conclude, cooling would seem to produce an increase in llama sperm chromatin decondensation. Also, 0.1% collagenase in H-TALP-BSA could be added to raw semen to aid its manipulation as it would not seem to increase DNA decondensation.

Epigenetic marks in zebrafish sperm: insights into chromatin compaction, maintenance of pluripotency, and the role of the paternal genome after fertilization

Epigenetic marks in zebrafish sperm: insights into chromatin compaction, maintenance of pluripotency, and the role of the paternal genome after fertilization

Male obesity and age in relationship to semen parameters and sperm chromatin integrity

Obesity can adversely affect human health, including fertility. While obesity can disturb the hormonal profile of the female organism and is associated with fertility loss, little is known about what effect male obesity has on fertility. The present study analysed sperm samples of 153 donors. The men were selected from couples attending an infertility clinic, who had tried for 12 months or more to achieve pregnancy without success. The age of the men under investigation was recorded, and their body mass index (BMI) was calculated. All semen samples were assessed for volume, concentration, motility and morphology. Sperm chromatin integrity was measured by sperm chromatin structure assay. Quality of sperm chromatin condensation was assessed by toluidine blue, aniline blue and chromomycin A3 staining. We can conclude that the impact of elevated BMI on the parameters investigated (basic semen parameters, chromatin integrity and chromatin condensation) was not proven in this study. On the other hand, ejaculate quality appeared to be affected by ageing. The impact was reflected by chromatin integrity, which is a factor that can substantially affect fertility in men, rather than by basic sperm parameters.

Sperm chromatin condensation in infertile men with varicocele before and after surgical repair

To assess sperm chromatin integrity in infertile men with varicocele before and after surgical repair. Prospective. Academic setting. Seventy-two infertile men with varicocele compared with 20 healthy fertile men. History taking, genital examination, semen analysis, sperm chromatin condensation assessment by aniline blue stain before and 3 months after varicocelectomy. Stained sperm heads (abnormal chromatin condensation) before and 3 months after varicocelectomy. The mean percentage of aniline blue-stained sperm heads was significantly higher in infertile men with varicocele compared with fertile controls. The mean percentage of stained sperm heads was significantly decreased in infertile men with varicocele 3 months after surgery compared with the preoperative data. There was a significant negative correlation between percentage of stained sperm heads and normal morphology where nonsignificant correlation was elicited regarding sperm count and sperm motility. There is a significant increase of abnormal sperm chromatin condensation in infertile men with varicocele that is markedly improved after varicocelectomy.