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Sample records for abnormal sperm chromatin

  1. Increased sperm ubiquitination correlates with abnormal chromatin integrity.

    PubMed

    Hodjat, M; Akhondi, M A; Al-Hasani, S; Mobaraki, M; Sadeghi, M R

    2008-09-01

    Ubiquitin, a 8.5 kDa peptide that marks other proteins for proteasomal degradation, tags defective spermatozoa during epididymal passage and is proposed as a biomarker for sperm quality. The present study was designed to evaluate the relationships between sperm ubiquitination, sperm chromatin integrity and semen parameters. Semen samples from 63 couples were collected and analysed according to World Health Organization criteria. Each sample was evaluated for sperm ubiquitination by the direct immunofluorescence method, using anti-ubiquitin antibodies. Chromatin integrity of the same samples was analysed using acridine orange (AO) and toluidine blue (TB) tests. A positive correlation was found between ubiquitinated spermatozoa and the percentage of spermatozoa with abnormal chromatin (AO: r = 0.58, P < 0.001 and TB: r = 0.48, P < 0.001). Negative correlations were obtained between sperm ubiquitination and: sperm count (r = -0.2, P = 0.048), sperm morphology (r = -0.36, P = 0.003), rapidly progressive motility (r = -0.25, P = 0.044) and slow progressive motility (r = -0.28, P = 0.022). Sperm ubiquitination was positively correlated with the percentage of immotile spermatozoa. These results show that among semen parameters, chromatin abnormality is more closely associated with sperm ubiquitination and further validate sperm ubiquitination as a suitable marker for sperm quality.

  2. Sperm Chromatin Integrity: Etiologies and Mechanisms of Abnormality, Assays, Clinical Importance, Preventing and Repairing Damage

    PubMed Central

    Hekmatdoost, Azita; Lakpour, Niknam; Sadeghi, Mohammad Reza

    2009-01-01

    The standard semen analysis is the first line and the most popular laboratory test in the diagnosis of male fertility. It evaluates sperm concentration, motility, morphology and their vitality. However, it is well-known that normal results of semen analysis can not exclude men from the causes of couples′ infertility. One of the most important parameters of sperm in its fertilizing potential is “Sperm chromatin integrity” that has direct positive correlation with Assisted Reproductive Techniques (ART) outcomes including; fertilization rate, embryo quality, pregnancy and successful delivery rate. It seems that sperm DNA chromatin integrity provides better diagnostic and prognostic approaches than standard semen parameters. For these reasons under-standing the sperm chromatin structure, etiology of sperm chromatin abnormality, identification factors that disturbs sperm chromatin integrity and the mechanism of their action can help in recognizing the causes of couples′ infertility. Various methods of its evaluation, its importance in male fertility, clinical relevance in the outcomes of ART and application of laboratory and medical protocols to improve this integrity have valuable position in diagnosis and treatment of male infertility. There has recently been interest in the subject and its application in the field of andrology. Therefore, with regard to the above mentioned importance of sperm chromatin integrity, this review article describes details of the useful information pertaining to sperm DNA damage including the origins, assessments, etiologies, clinical aspects, and prevention of it. PMID:23408441

  3. Deficiency of the multi-copy mouse Y gene Sly causes sperm DNA damage and abnormal chromatin packaging.

    PubMed

    Riel, Jonathan M; Yamauchi, Yasuhiro; Sugawara, Atsushi; Li, Ho Yan J; Ruthig, Victor; Stoytcheva, Zoia; Ellis, Peter J I; Cocquet, Julie; Ward, Monika A

    2013-02-01

    In mouse and man Y chromosome deletions are frequently associated with spermatogenic defects. Mice with extensive deletions of non-pairing Y chromosome long arm (NPYq) are infertile and produce sperm with grossly misshapen heads, abnormal chromatin packaging and DNA damage. The NPYq-encoded multi-copy gene Sly controls the expression of sex chromosome genes after meiosis and Sly deficiency results in a remarkable upregulation of sex chromosome genes. Sly deficiency has been shown to be the underlying cause of the sperm head anomalies and infertility associated with NPYq gene loss, but it was not known whether it recapitulates sperm DNA damage phenotype. We produced and examined mice with transgenically (RNAi) silenced Sly and demonstrated that these mice have increased incidence of sperm with DNA damage and poorly condensed and insufficiently protaminated chromatin. We also investigated the contribution of each of the two Sly-encoded transcript variants and noted that the phenotype was only observed when both variants were knocked down, and that the phenotype was intermediate in severity compared with mice with severe NPYq deficiency. Our data demonstrate that Sly deficiency is responsible for the sperm DNA damage/chromatin packaging defects observed in mice with NPYq deletions and point to SLY proteins involvement in chromatin reprogramming during spermiogenesis, probably through their effect on the post-meiotic expression of spermiogenic genes. Considering the importance of the sperm epigenome for embryonic and fetal development and the possibility of its inter-generational transmission, our results are important for future investigations of the molecular mechanisms of this biologically and clinically important process.

  4. Toxicants and human sperm chromatin integrity.

    PubMed

    Delbès, Geraldine; Hales, Barbara F; Robaire, Bernard

    2010-01-01

    The integrity of the paternal genome is essential as the spermatozoon can bring genetic damage into the oocyte at fertilization and contribute to the development of abnormal pregnancy outcome. During the past two decades, many assays have been developed to measure sperm DNA strand breaks, chromatin structure and compaction and assess the proteins associated with the DNA, as well as epigenetic modifications. Using these assays, it has been shown that exposure to physical agents or chemicals, including therapeutic drugs and environmental toxicants, can affect the integrity of sperm chromatin, inducing structural, genetic and/or epigenetic abnormalities. The mechanisms by which such damage is triggered are still largely unresolved and the susceptibility of each individual will depend on their genetic background, lifestyle and exposure to various insults. Depending on the nature of the chemicals, they may directly target the DNA, induce an oxidative stress, or modify the epigenetic elements. The significance of measuring the sperm chromatin integrity comes from the fact that this end-point correlates well with the low IVF and ICSI outcomes, and idiopathic infertility. Nevertheless, it is hard to establish a direct link between the paternal sperm chromatin integrity and the health of the future generations. Thus, it seems essential to undertake studies that will resolve the impact of chemical and environmental factors on chromatin structure and epigenetic components of human spermatozoa and to elucidate what sperm nuclear end-points are predictors of the quality of progeny outcome.

  5. Human sperm chromatin stabilization: a proposed model including zinc bridges.

    PubMed

    Björndahl, Lars; Kvist, Ulrik

    2010-01-01

    The primary focus of this review is to challenge the current concepts on sperm chromatin stability. The observations (i) that zinc depletion at ejaculation allows a rapid and total sperm chromatin decondensation without the addition of exogenous disulfide cleaving agents and (ii) that the human sperm chromatin contains one zinc for every protamine for every turn of the DNA helix suggest an alternative model for sperm chromatin structure may be plausible. An alternative model is therefore proposed, that the human spermatozoon could at ejaculation have a rapidly reversible zinc dependent chromatin stability: Zn(2+) stabilizes the structure and prevents the formation of excess disulfide bridges by a single mechanism, the formation of zinc bridges with protamine thiols of cysteine and potentially imidazole groups of histidine. Extraction of zinc enables two biologically totally different outcomes: immediate decondensation if chromatin fibers are concomitantly induced to repel (e.g. by phosphorylation in the ooplasm); otherwise freed thiols become committed into disulfide bridges creating a superstabilized chromatin. Spermatozoa in the zinc rich prostatic fluid (normally the first expelled ejaculate fraction) represent the physiological situation. Extraction of chromatin zinc can be accomplished by the seminal vesicular fluid. Collection of the ejaculate in one single container causes abnormal contact between spermatozoa and seminal vesicular fluid affecting the sperm chromatin stability. There are men in infertile couples with low content of sperm chromatin zinc due to loss of zinc during ejaculation and liquefaction. Tests for sperm DNA integrity may give false negative results due to decreased access for the assay to the DNA in superstabilized chromatin.

  6. Sperm chromatin structure assay (SCSA®).

    PubMed

    Evenson, Donald P

    2013-01-01

    The SCSA(®) is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA(®) is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) on a 1,024 × 1,024 channel scale, not only on DNA fragmentation but also on abnormal sperm characterized by lack of normal exchange of histones to protamines. Raw semen/sperm aliquots or purified sperm can be flash frozen, placed in a box with dry ice and shipped by overnight courier to an experienced SCSA(®) lab. The samples are individually thawed, prepared, and analyzed in ∼10 min. Of significance, data on 5,000 individual sperm are recorded on a 1,024 × 1,024 dot plot of green (native DNA) and red (broken DNA) fluorescence. Repeat measurements have virtually identical dot plot patterns demonstrating that the low pH treatment that opens up the DNA strands at the sites of breaks and staining by acridine orange (AO) are highly precise and repeatable (CVs of 1-3%) and the same between fresh and frozen samples. SCSAsoft(®) software transforms the X-Y data to total DNA stainability versus red/red + green fluoresence (DFI) providing a more accurate determination of % DFI as well as the more sensitive value of standard deviation of DFI (SD DFI) as demonstrated by animal fertility and dose-response toxicology studies. The current established clinical threshold is 25% DFI for placing a man into a statistical probability of the following: (a) longer time to natural pregnancy, (b) low odds of IUI pregnancy, (c) more miscarriages, or (d) no pregnancy. Changes in lifestyle as well as medical intervention can lower the %DFI to increase the probability of natural pregnancy. Couples of men with >25% DFI are counseled to try ICSI and when in the >50% range may consider TESE/ICSI. The SCSA(®) simultaneously determines the % of sperm with high DNA stainability (%HDS

  7. Chromosomal abnormalities in human sperm

    SciTech Connect

    Martin, R.H.

    1985-01-01

    The ability to analyze human sperm chromosome complements after penetration of zona pellucida-free hamster eggs provides the first opportunity to study the frequency and type of chromosomal abnormalities in human gametes. Two large-scale studies have provided information on normal men. We have studied 1,426 sperm complements from 45 normal men and found an abnormality rate of 8.9%. Brandriff et al. (5) found 8.1% abnormal complements in 909 sperm from 4 men. The distribution of numerical and structural abnormalities was markedly dissimilar in the 2 studies. The frequency of aneuploidy was 5% in our sample and only 1.6% in Brandriff's, perhaps reflecting individual variability among donors. The frequency of 24,YY sperm was low: 0/1,426 and 1/909. This suggests that the estimates of nondisjunction based on fluorescent Y body data (1% to 5%) are not accurate. We have also studied men at increased risk of sperm chromosomal abnormalities. The frequency of chromosomally unbalanced sperm in 6 men heterozygous for structural abnormalities varied dramatically: 77% for t11;22, 32% for t6;14, 19% for t5;18, 13% for t14;21, and 0% for inv 3 and 7. We have also studied 13 cancer patients before and after radiotherapy and demonstrated a significant dose-dependent increase of sperm chromosome abnormalities (numerical and structural) 36 months after radiation treatment.

  8. APPLICATION OF THE SPERM CHROMATIN STRUCTURE ASSAY TO THE TEPLICE PROGRAM SEMEN STUDIES: A NEW METHOD FOR EVALUATING SPERM NUCLEAR CHROMATIN DAMAGE

    EPA Science Inventory

    ABSTRACT
    A measure of sperm chromatin integrity was added to the routine semen end points evaluated in the Teplice Program male reproductive health studies. To address the hypothesis that exposure to periods of elevated air pollution may be associated with abnormalities in sp...

  9. Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa.

    PubMed

    Franco, J G; Mauri, A L; Petersen, C G; Massaro, F C; Silva, L F I; Felipe, V; Cavagna, M; Pontes, A; Baruffi, R L R; Oliveira, J B A; Vagnini, L D

    2012-02-01

    The aim of this investigation was to determine the presence of abnormal sperm chromatin packaging in spermatozoa with large nuclear vacuoles (LNV) selected via high magnification by analysing the pattern of chromomycin A3 (CMA3) staining. A prospective observational study was designed to analyse semen samples obtained from 66 men undergoing infertility diagnosis and treatment. The numbers of cells with normal (dull yellow staining of the sperm head/CMA3-negative) and abnormal (bright yellow fluorescence of the sperm head/CMA3-positive) chromatin packaging were determined on slides with normal and LNV spermatozoa. The presence of bright yellow fluorescence (CMA3-positive) was significantly higher (p < 0.0001) in spermatozoa with LNV than in normal spermatozoa (719/1351; 53.2% vs. 337/835; 40.3%, respectively), reflecting a higher percentage of abnormal chromatin packaging in spermatozoa with large LNV. Our data support the hypothesis that the presence of LNV reflects the presence of abnormal chromatin packaging, which may facilitate sperm DNA damage. As sperm nuclear vacuoles are evaluated more precisely at high magnifications using motile sperm organelle morphology examination (MSOME), the present results support the use of high-magnification sperm selection for intracytoplasmic sperm injection (ICSI).

  10. Quantitative evaluation of radiation-induced changes in sperm morphology and chromatin distribution

    SciTech Connect

    Aubele, M.; Juetting, U.R.; Rodenacker, K.; Gais, P.; Burger, G.; Hacker-Klom, U. )

    1990-01-01

    Sperm head cytometry provides a useful assay for the detection of radiation-induced damage in mouse germ cells. Exposure of the gonads to radiation is known to lead to an increase of diploid and higher polyploid sperm and of sperm with head shape abnormalities. In the pilot studies reported here quantitative analysis of the total DNA content, the morphology, and the chromatin distribution of mouse sperm was performed. The goal was to evaluate the discriminative power of features derived by high resolution image cytometry in distinguishing sperm of control and irradiated mice. Our results suggest that besides the induction of the above mentioned variations in DNA content and shape of sperm head, changes of the nonhomogeneous chromatin distribution within the sperm may also be used to quantify the radiation effect on sperm cells. Whereas the chromatin distribution features show larger variations for sperm 21 days after exposure (dpr), the shape parameters seem to be more important to discriminate sperm 35 dpr. This may be explained by differentiation processes, which take place in different stages during mouse spermatogenesis.

  11. Altered sperm chromatin structure in mice exposed to sodium fluoride through drinking water.

    PubMed

    Sun, Zilong; Niu, Ruiyan; Wang, Bin; Wang, Jundong

    2014-06-01

    This study investigated the effects of sodium fluoride (NaF) on sperm abnormality, sperm chromatin structure, protamine 1 and protamine 2 (P1 and P2) mRNA expression, and histones expression in sperm in male mice. NaF was orally administrated to male mice at 30, 70, and 150 mg/l for 49 days (more than one spermatogenic cycle). Sperm head and tail abnormalities were significantly enhanced at middle and high doses. Similarly, sperm chromatin structure was also adversely affected by NaF exposure, indicating DNA integrity damage. Furthermore, middle and high NaF significantly reduced the mRNA expressions of P1 and P2, and P1/P2 ratio, whereas the sperm histones level was increased, suggesting the abnormal histone-protamine replacement. Therefore, we concluded that the mechanism by which F induced mice sperm abnormality and DNA integrity damage may involved in the alterations in P1, P2, and histones expression in sperm of mice.

  12. Advancing age increases sperm chromatin damage and impairs fertility in peroxiredoxin 6 null mice

    PubMed Central

    Ozkosem, Burak; Feinstein, Sheldon I.; Fisher, Aron B.; O’Flaherty, Cristian

    2015-01-01

    Due to socioeconomic factors, more couples are choosing to delay conception than ever. Increasing average maternal and paternal age in developed countries over the past 40 years has raised the question of how aging affects reproductive success of males and females. Since oxidative stress in the male reproductive tract increases with age, we investigated the impact of advanced paternal age on the integrity of sperm nucleus and reproductive success of males by using a Prdx6−/− mouse model. We compared sperm motility, cytoplasmic droplet retention sperm chromatin quality and reproductive outcomes of young (2-month-old), adult (8-month-old), and old (20-month-old) Prdx6−/− males with their age-matched wild type (WT) controls. Absence of PRDX6 caused age-dependent impairment of sperm motility and sperm maturation and increased sperm DNA fragmentation and oxidation as well as decreased sperm DNA compaction and protamination. Litter size, total number of litters and total number of pups per male were significantly lower in Prdx6−/− males compared to WT controls. These abnormal reproductive outcomes were severely affected by age in Prdx6−/− males. In conclusion, the advanced paternal age affects sperm chromatin integrity and fertility more severely in the absence of PRDX6, suggesting a protective role of PRDX6 in age-associated decline in the sperm quality and fertility in mice. PMID:25796034

  13. Chd5 orchestrates chromatin remodeling during sperm development

    PubMed Central

    Li, Wangzhi; Wu, Jie; Kim, Sang-Yong; Zhao, Ming; Hearn, Stephen A.; Zhang, Michael Q.; Meistrich, Marvin L.

    2014-01-01

    One of the most remarkable chromatin remodeling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodeling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orchestrates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction, and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone-to-protamine replacement and depict the cascade of molecular events underlying chromatin remodeling during this process of extensive chromatin remodeling. PMID:24818823

  14. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation.

    PubMed

    Fernández, Jose Luis; Muriel, Lourdes; Rivero, Maria Teresa; Goyanes, Vicente; Vazquez, Rosana; Alvarez, Juan G

    2003-01-01

    Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test

  15. The relationship between sperm morphology and chromatin integrity in the koala (Phascolarctos cinereus) as assessed by the Sperm Chromatin Dispersion test (SCDt).

    PubMed

    Johnston, Stephen D; López-Fernández, Carmen; Gosálbez, Altea; Zee, Yengpeng; Holt, William V; Allen, Camryn; Gosálvez, Jaime

    2007-01-01

    that is damaged as part of the normal processing of the spermatozoa and is a consequence of the lack of cysteine residues and associated stabilizing disulfide bonds in marsupial sperm DNA. "True" sperm DNA damage is most likely associated with KSM-3, which shows a massive dispersion of chromatin similar to that described in other species. A model of koala sperm chromatin structure is proposed to explain the behavior of the sperm nuclei after the SCDt. Further studies are required to determine whether DNA damage found in KSM-2 is indicative of single-stranded DNA breakage associated with an inherent lack of cysteine residues in marsupial sperm chromatin. Conversely, it will also be important to establish whether KSM-3 is caused by an increased incidence of double-stranded DNA breakage and whether this abnormality is correlated with impaired fertility as it is in other species.

  16. Investigation of the association between the outcomes of sperm chromatin condensation and decondensation tests, and assisted reproduction techniques.

    PubMed

    Irez, T; Sahmay, S; Ocal, P; Goymen, A; Senol, H; Erol, N; Kaleli, S; Guralp, O

    2015-05-01

    The main purpose of this prospective study is to examine possible influences of abnormalities of sperm nuclear condensation and chromatin decondensation with sodium dodecyl sulphate (SDS)-EDTA on outcomes of intrauterine insemination (IUI) or intracytoplasmic sperm injection (ICSI) cycles. Semen samples from 122 IUI and 236 ICSI cycles were evaluated. Before semen preparation for IUI or ICSI, basic semen analysis was performed and a small portion from each sample was spared for fixation. The condensation of sperm nuclear chromatin was evaluated with acidic aniline blue, followed by sperm chromatin decondensation by SDS-EDTA and evaluation under light microscope. Ongoing pregnancy rate was 24% and 26.2% in the IUI and ICSI groups respectively. The chromatin condensation rate was significantly higher in the ongoing pregnancy-positive group compared to the negative group, both in IUI (P = 0.042) and ICSI groups (P = 0.027), and it was positively correlated with ongoing pregnancy rate in both IUI and ICSI groups (P = 0.015, r = 0.214 and P = 0.014, r = 0.312 respectively). Chromatin decondensation rates were not significantly different in neither of the groups. These results indicate that IUI and ICSI outcome is influenced by the rate of spermatozoa with abnormal chromatin condensation. Sperm chromatin condensation with aniline blue is useful for selecting assisted reproduction techniques (ART) patients.

  17. Recent knowledge concerning mammalian sperm chromatin organization and its potential weaknesses when facing oxidative challenge.

    PubMed

    Noblanc, Anais; Kocer, Ayhan; Drevet, Joël R

    2014-01-01

    Spermatozoa are the smallest and most cyto-differentiated mammalian cells. From a somatic cell-like appearance at the beginning of spermatogenesis, the male germ cell goes through a highly sophisticated process to reach its final organization entirely devoted to its mission which is to deliver the paternal genome to the oocyte. In order to fit the paternal DNA into the tiny spermatozoa head, complete chromatin remodeling is necessary. This review essentially focuses on present knowledge of this mammalian sperm nucleus compaction program. Particular attention is given to most recent advances that concern the specific organization of mammalian sperm chromatin and its potential weaknesses. Emphasis is placed on sperm DNA oxidative damage that may have dramatic consequences including infertility, abnormal embryonic development and the risk of transmission to descendants of an altered paternal genome.

  18. Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells.

    PubMed

    Huser, Thomas; Orme, Christine A; Hollars, Christopher W; Corzett, Michele H; Balhorn, Rod

    2009-05-01

    Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

  19. Raman Spectroscopy of DNA Packaging in Individual Human Sperm Cells distinguishes Normal from Abnormal Cells

    SciTech Connect

    Huser, T; Orme, C; Hollars, C; Corzett, M; Balhorn, R

    2009-03-09

    Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

  20. Combination of density gradient centrifugation and swim-up methods effectively decreases morphologically abnormal sperms

    PubMed Central

    YAMANAKA, Masaya; TOMITA, Kazuhisa; HASHIMOTO, Shu; MATSUMOTO, Hiroshi; SATOH, Manabu; KATO, Hiromi; HOSOI, Yoshihiko; INOUE, Masayasu; NAKAOKA, Yoshiharu; MORIMOTO, Yoshiharu

    2016-01-01

    Density gradient centrifugation (DGC) and swim-up techniques have been reported for semen preparation in assisted reproductive techniques in humans. We investigated whether semen preparation using a combination of DGC and swim-up techniques could effectively decrease morphologically abnormal human sperms at the ultrastructural level. Semen samples were obtained from 16 infertile males and fractionated by swim-up following DGC. Ultrastructural abnormalities of sperms obtained from original semen, lower layer of swim-up following DGC, and upper layer of swim-up following DGC were analyzed by transmission electron microscopy. The correlation among ultrastructural head abnormality in sperms from the upper layer of swim-up, fertilization in in vitro fertilization, and pregnancy after embryo transfer was also investigated. Furthermore, sperms with DNA fragmentation in the samples processed via a combination of DGC and swim-up was assessed in a sperm chromatin structure assay. Ultrastructural abnormalities in sperm heads and tails in the upper layer after swim-up following DGC was the lowest among the three groups. Sperms with nuclear vacuoles were the most difficult to eliminate using a combination of DGC and swim-up in all types of head abnormalities. A negative correlation was confirmed between the fertilization rates of intracytoplasmic sperm injection and head abnormality of sperms obtained from the upper layer of the swim-up following DGC. Sperms with DNA fragmentation were effectively decreased using the combination of two techniques. In conclusion, the combination of DGC and swim-up effectively decreased the number of sperms with ultrastructural abnormalities both in the head and in the tail. However, sperms with ultrastructural abnormalities that cannot be completely decreased using a combination of DGC and swim-up may impair fertilization in some cases of intracytoplasmic sperm injection. PMID:27616283

  1. Proteomics and the genetics of sperm chromatin condensation

    PubMed Central

    Oliva, Rafael; Castillo, Judit

    2011-01-01

    Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell. Proteomics allows the identification of the proteins that compose the spermatogenic cells and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and to study the sperm proteins. Catalogs of thousands of testis and spermatozoan proteins in human and different model species are becoming available, setting up the basis for subsequent research, diagnostic applications and possibly the future development of specific treatments. The present review intends to summarize the key genetic and chromatin changes at the different stages of spermatogenesis and in the mature sperm cell and to comment on the presently available proteomic studies. PMID:21042303

  2. Chemical induction of sperm abnormalities in mice.

    PubMed Central

    Wyrobek, A J; Bruce, W R

    1975-01-01

    The sperm of (C57BL X C3H)F1 mice were examined 1, 4, and 10 weeks after a subacute treatment with one of 25 chemicals at two or more dose levels. The fraction of sperm that were abnormal in shape was elevated above control values of 1.2-3.4% for methyl methanesulfonate, ethyl methanesulfonate, griseofulvin, benzo[a]pyrene, METEPA [tris(2-methyl-l-aziridinyl)phosphine oxide], THIO-TEPA [tris(l-aziridinyl)phosphine sulfide], mitomycin C, myleran, vinblastine sulphate, hydroxyurea, 3-methylcholanthrene, colchicine, actinomycin D, imuran, cyclophosphamide, 5-iododeoxyuridine, dichlorvos, aminopterin, and trimethylphosphate. Dimethylnitrosamine, urethane, DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane], 1,1-dimethylhydrazine, caffeine, and calcium cyclamate did not induce elevated levels of sperm abnormalities. The results suggest that sperm abnormalities might provide a rapid inexpensive mammalian screen for agents that lead to errors in the differentiation of spermatogenic stem cells in vivo and thus indicate agents which might prove to be mutagenic, teratogenic, or carcinogenic. Images PMID:1060122

  3. Acute and chronic effects of gold nanoparticles on sperm parameters and chromatin structure in Mice

    PubMed Central

    Nazar, Mahsa; Talebi, Ali Reza; Hosseini Sharifabad, Mohammad; Abbasi, Abolghasem; Khoradmehr, Arezoo; Danafar, Amir Hossein

    2016-01-01

    Background: The particles in the range of 1-100 nm are called nanoparticles. Gold nanoparticle is one of the most important metal nanoparticles with wide usage. Objective: This study investigated the effects of gold nanoparticles on sperm parameters and chromatin structure in mice. Materials and Methods: In this experimental study, 72 male bulb-c mice were divided into 9 groups including: 4 Sham groups (Sc 1-4), 4 experimental groups (Au 1-4), and 1 control group (C). Experimental groups received 40 and 200 µg/kg/day soluble gold (Au) nano-particles for 7 and 35 days, by intra peritoneal injection, respectively. Sham groups were treated with 1.2 mM sodium citrate solution with 40 and 200 µg/kg/day doses for same days and control group did not receive any materials. Motility and Morphology of spermatozoa were analyzed. Chromatin quality was also evaluated using AB (Aniline blue), TB (Toluidine blue) and CMA3 (Chromomycin A3) staining methods. Results: The sperm analysis results showed that motility and morphology of sperm in experimental groups (especially in groups that have been treated for 35 days with nano-particles) had significant decrease in comparison with control group. TB, AB and CMA3 results showed a significant increase in abnormal spermatozoa from all Au-treated groups. Conclusion: Gold nano-particles firstly can reduce the sperm parameters such as motility and normal morphology and secondly affect sperm chromatin remodeling and cause the increase instability of chromatin and also increase the rate of sperm DNA damage. These deleterious effects were more obvious in maximum dose and chronic phase. PMID:27921087

  4. Long-term effects of triethylenemelamine exposure on mouse testis cells and sperm chromatin structure assayed by flow cytometry

    SciTech Connect

    Evenson, D.P.; Baer, R.K.; Jost, L.K. )

    1989-01-01

    The toxic and potentially mutagenic actions of triethylenemelamine (TEM) on mouse body and testis weights, testicular cell kinetics, sperm production, sperm head morphology, and sperm chromatin structure were assessed in two experiments. The first experiment examined effects of four dose levels of TEM, assayed 1, 4, or 10 wk after toxic exposure. In the second study, effects from five dosage levels were measured at 1, 4, and 10 wk, and the highest dosage level was evaluated over 44 wk. TEM produced an expected dose related loss of spermatogenic activity and subsequent recovery as determined by dual-parameter (DNA, RNA) flow cytometry (FCM) measurements of testicular cells. Both testicular weights and caudal sperm reserves remained generally below controls after 44 wk recovery following exposure to the highest dosage. Chromatin structure alterations, defined as increased susceptibility to DNA denaturation in situ, and sperm head morphology were highly correlated with dose and with each other. Sperm head morphology and sperm chromatic structure remained abnormal at 44 wk for the 1.0 mg/kg TEM dosage, suggesting that the abnormalities, present long after the initial toxic response, may be a result of mutation. This study demonstrates that flow cytometry provides a unique, rapid, and efficient means to measure effects of reproductive toxins and potential mutagens.

  5. Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA[reg])

    SciTech Connect

    Evenson, Donald P. . E-mail: scsa@brookings.net; Wixon, Regina

    2005-09-01

    Studies over the past two decades have clearly shown that reproductive toxicants cause sperm DNA fragmentation. This DNA fragmentation can usually be detected prior to observing alterations of metaphase chromosomes in embryos. Thus, Sperm Chromatin Structure Assay (SCSA)-detected DNA damage is viewed as the molecular precursor to later gross chromosome damage observed under the light microscope. SCSA measurements of animal or human sperm consist of first obtaining a fresh or flash frozen neat semen sample in LN2 or dry ice. Samples are then sent to a SCSA diagnostic laboratory where the samples are thawed, diluted to {approx}1-2 x 106 sperm/ml, treated for 30 s with a pH 1.2 detergent buffer and then stained with acridine orange (AO). The low pH partially denatures DNA at the sites of DNA strand breaks and the AO-ssDNA fluoresces red while the AO-dsDNA fluoresces green. Flow cytometry measurements of 5000 sperm/sample provide statistically robust data on the ratio of red to green sperm, the extent of the DNA fragmentation and the standard deviations of measures. Numerous experiments on rodents treated with reproductive toxicants clearly showed that SCSA measures are highly dose responsive and have a very low CV. Different agents that act on germ cells at various stages of development usually showed sperm DNA fragmentation when that germ cell fraction arrived in the epididymis or ejaculate. Some of these treated samples were capable of successful in vitro fertilization but with frequent embryo failure. A 2-year longitudinal study of men living a valley town with a reported abnormal level of infertility and spontaneous miscarriages and also a seasonal atmospheric smog pollution, showed, for the first time, that SCSA measurements of human sperm DNA fragmentation were detectable and correlated with dosage of air pollution while the classical semen measures were not correlated. Also, young men spraying pesticides without protective gear are at an increased risk for

  6. Effects of X-irradiation on mouse testicular cells and sperm chromatin structure

    SciTech Connect

    Sailer, B.L.; Jost, L.K.; Erickson, K.R.; Tajiran, M.A.; Evenson, D.P.

    1995-07-01

    The testicular regions of male mice were exposed to x-ray doses ranging from 0 to 400 rads. Forty days after exposure the mice were killed and the testes and cauda epididymal sperm removed surgically. Flow cytometric measurements of acridine orange stained testicular samples indicated a repopulation of testicular samples indicated a repopulation of testicular cell types following x-ray killing of stem cells. Cauda epididymal sperm were analyzed by the sperm chromatin structure assay (SCSA), a flow cytometric measurement of the susceptibility of the sperm nuclear DNA to in situ acid denaturation. The SCSA detected increased susceptibility to DNA denaturation in situ after 12.5 rads of x-ray exposure, with significant increases following 25 rads. Abnormal sperm head morphology was not significantly increased until the testes were exposed to 60 rads of x-rays. These data suggest that the SCSA is currently the most sensitive, noninvasive method of detecting x-ray damage to testicular stem spermatogonia. 47 refs., 5 figs.

  7. Correlation between sperm ultrastructure in infertile patients with abnormal sperm morphology and DNA damage.

    PubMed

    He, M; Tan, L

    2015-12-15

    This study explored the correlation between sperm ultrastructure in infertile patients with abnormal sperm morphology and DNA damage. Three unusual sperm morphologies were selected for the experimental group namely case 1 (95% headless sperm), case 2 (98% headless sperm), and case 3 (100% headless sperm), and the control group consisted of 2 subjects (20 and 15% headless sperm). For case 1, the patient was negative for sexually transmitted diseases and had normal semen plasma biochemistry, reproductive hormones, peripheral blood chromosomes, and azoospermia factor (AZF). The aneuploid rate of sperm chromosomes was 0.6%, and DNA damage index of sperm nuclei was 84.4%. The partner of this patient did not get pregnant after artificial reproductive technology assistance. For case 2, the aneuploid rate of sperm chromosomes was 0.8% and DNA damage index of sperm nuclei was 95%. This patient and his spouse did not choose assisted reproduction. For case 3, reproductive hormones, peripheral blood chromosomes and AZF were normal and the aneuploid rate of sperm chromosomes was 0.2%. The wife of this patient gave birth to a healthy baby after ova removal, fertilization and transplantation. For the control group, the aneuploid rate of sperm chromosomes and DNA damage index of sperm nuclei were approximately 0.3 and 30%, respectively. To sum up, sperm ultrastructure of infertile patients suffering from unusual sperm morphology is associated with DNA damage to some extent and can cause infertility. However, pregnancy is still possible through intracytoplasmic sperm injection.

  8. Assessment of sperm DNA fragmentation in stallion (Equus caballus) and donkey (Equus asinus) using the sperm chromatin dispersion test.

    PubMed

    Cortés-Gutiérrez, E I; Crespo, F; Serres-Dalmau, C; Gutiérrez de las Rozas, A L; Dávila-Rodríguez, M I; López-Fernández, C; Gósalvez, J

    2009-10-01

    Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present. Only the SCD test, originally developed for stallions, produced stable and consistent results, and was deemed suitable to assess DNA fragmentation in sperm samples from donkeys. Image analysis was used to compare differences between the SCD methodology applied to stallion and donkey semen samples processed under the same experimental conditions. The extent of SCD in the SCD test was approximately 20% lower in donkey sperm than in stallion sperm. Yet, the ratio of chromatin sperm dispersion achieved in fragmented and unfragmented nuclei did not differ significantly between species. These data suggest that a similar protein depletion treatment can cause differences in protein removal in equivalent cells from different species and that sperm chromatin may be organized differently in stallions and donkeys.

  9. Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test.

    PubMed

    Carretero, M I; Lombardo, D; Arraztoa, C C; Giuliano, S M; Gambarotta, M C; Neild, D M

    2012-03-01

    The integrity of sperm chromatin is now viewed as an important factor in male fertility and in early embryonic development. The objectives of this study were: (1) adapt the simple and inexpensive sperm chromatin dispersion (SCD) test to evaluate DNA fragmentation in llama sperm and establish the halo patterns observed in this species, (2) determine an effective and reliable positive control for this technique and (3) evaluate correlation between the SCD test and the toluidine blue (TB) stain. To adapt the SCD test, three different mercaptoethanol (ME) concentrations were assayed (2.5%, 5% and 10% ME). To determine an effective positive control, three treatments (incubation at 100 °C for 30 min, incubation with 0.3 M NaOH for 30 min at room temperature and exposure to UV light for 2h) were assayed. The concentration selected to use in the SCD test was 5% ME, because it produced the largest halo while still conserving the structure of the core. Four DNA dispersion patterns were clearly observed: (I) nuclei with large DNA dispersion halos; (II) nuclei with medium halos; (III) nuclei with very small halos and (IV) nuclei with no halo. All treatments used as positive controls were effective in producing DNA fragmentation. A high correlation (r=0.84, P=0.03) was observed between spermatozoa without halos and TB positive cells. To conclude, SCD patterns in llama sperm have been established as well as a repeatable positive control for the assay. The SCD test and TB stain are simple and inexpensive techniques that can be used to evaluate DNA damage in llama sperm.

  10. Incidence, structure and morphological classification of abnormal sperm in the emu (Dromaius novaehollandiae).

    PubMed

    du Plessis, Lizette; Soley, John T

    2011-03-01

    Little detailed information is currently available on the incidence and morphological characteristics of abnormal sperm in the emu (Dromaius novaehollandiae) and of ratites in general. This situation is further compounded by the lack of a uniform system for the morphological classification of avian sperm defects. Considering the important role that sperm morphology plays in the assessment of semen quality, a detailed description of avian sperm defects is of paramount importance. Based on morphological data provided by light and electron microscopy, a mean of 17.3% abnormal sperm was recorded in semen samples collected from the distal deferent duct of four adult emus during the middle of the breeding season. Four categories of defects were identified. Head defects (57.2% of total defects) consisted of bent heads, macrocephalic heads, round heads and acephalic sperm. Zones of incomplete chromatin condensation and retained cytoplasmic droplets appeared to be implicated in head bending, while giant heads were often associated with multiple tails. Acephalic sperm revealed a complete tail devoid of a head which was replaced by a small spherical structure. Tail defects (22.6% of total defects) were subdivided into neck/midpiece defects and principal piece defects. In the neck/midpiece region disjointed sperm were the exclusive defect noted and were characterized by the complete separation of the head and midpiece in the neck region but within the confines of the plasmalemma. Defects observed in the principal piece were subdivided into short tails, coiled tails and multiple tails. No conclusive evidence was obtained that tail coiling represented the 'Dag' defect. Biflagellate sperm were the most common form of multiple tails, demonstrating two complete tails with all the normal structural elements. Cytoplasmic droplets (13.9% of total defects) were classified as a separate defect. The location and eccentric positioning of retained cytoplasmic droplets was similar to that

  11. The sperm nucleus: chromatin, RNA and the nuclear matrix

    PubMed Central

    Johnson, Graham D.; Lalancette, Claudia; Linnemann, Amelia K.; Leduc, Frédéric; Boissonneault, Guylain; Krawetz, Stephen A.

    2017-01-01

    Within the sperm nucleus the paternal genome remains functionally inert and protected following protamination. This is marked by a structural morphogenesis that is heralded by a striking reduction in nuclear volume. Despite these changes, both human and mouse spermatozoa maintain low levels of nucleosomes that appear non-randomly distributed throughout the genome. These regions may be necessary for organizing higher order genomic structure through interactions with the nuclear matrix. The promoters of this transcriptionally quiescent genome are differentially marked by modified histones that may poise downstream epigenetic effects. This notion is supported by increasing evidence that the embryo inherits these differing levels of chromatin organization. In concert with the suite of RNAs retained in the mature sperm they may synergistically interact to direct early embryonic gene expression. Irrespective, these features reflect the transcriptional history of spermatogenic differentiation. As such they may soon be utilized as clinical markers of male fertility. In this review we explore and discuss how this may be orchestrated. PMID:20876223

  12. Protection of cisplatin-induced spermatotoxicity, DNA damage and chromatin abnormality by selenium nano-particles

    SciTech Connect

    Rezvanfar, Mohammad Amin; Rezvanfar, Mohammad Ali; Shahverdi, Ahmad Reza; Ahmadi, Abbas; Baeeri, Maryam; Mohammadirad, Azadeh; Abdollahi, Mohammad

    2013-02-01

    Cisplatin (CIS), an anticancer alkylating agent, induces DNA adducts and effectively cross links the DNA strands and so affects spermatozoa as a male reproductive toxicant. The present study investigated the cellular/biochemical mechanisms underlying possible protective effect of selenium nano-particles (Nano-Se) as an established strong antioxidant with more bioavailability and less toxicity, on reproductive toxicity of CIS by assessment of sperm characteristics, sperm DNA integrity, chromatin quality and spermatogenic disorders. To determine the role of oxidative stress (OS) in the pathogenesis of CIS gonadotoxicity, the level of lipid peroxidation (LPO), antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and peroxynitrite (ONOO) as a marker of nitrosative stress (NS) and testosterone (T) concentration as a biomarker of testicular function were measured in the blood and testes. Thirty-two male Wistar rats were equally divided into four groups. A single IP dose of CIS (7 mg/kg) and protective dose of Nano-Se (2 mg/kg/day) were administered alone or in combination. The CIS-exposed rats showed a significant increase in testicular and serum LPO and ONOO level, along with a significant decrease in enzymatic antioxidants levels, diminished serum T concentration and abnormal histologic findings with impaired sperm quality associated with increased DNA damage and decreased chromatin quality. Coadministration of Nano-Se significantly improved the serum T, sperm quality, and spermatogenesis and reduced CIS-induced free radical toxic stress and spermatic DNA damage. In conclusion, the current study demonstrated that Nano-Se may be useful to prevent CIS-induced gonadotoxicity through its antioxidant potential. Highlights: ► Cisplatin (CIS) affects spermatozoa as a male reproductive toxicant. ► Effect of Nano-Se on CIS-induced spermatotoxicity was investigated. ► CIS-exposure induces oxidative sperm DNA damage

  13. Unlocking sperm chromatin at fertilization requires a dedicated egg thioredoxin in Drosophila

    PubMed Central

    Tirmarche, Samantha; Kimura, Shuhei; Dubruille, Raphaëlle; Horard, Béatrice; Loppin, Benjamin

    2016-01-01

    In most animals, the extreme compaction of sperm DNA is achieved after the massive replacement of histones with sperm nuclear basic proteins (SNBPs), such as protamines. In some species, the ultracompact sperm chromatin is stabilized by a network of disulfide bonds connecting cysteine residues present in SNBPs. Studies in mammals have established that the reduction of these disulfide crosslinks at fertilization is required for sperm nuclear decondensation and the formation of the male pronucleus. Here, we show that the Drosophila maternal thioredoxin Deadhead (DHD) is specifically required to unlock sperm chromatin at fertilization. In dhd mutant eggs, the sperm nucleus fails to decondense and the replacement of SNBPs with maternally-provided histones is severely delayed, thus preventing the participation of paternal chromosomes in embryo development. We demonstrate that DHD localizes to the sperm nucleus to reduce its disulfide targets and is then rapidly degraded after fertilization. PMID:27876811

  14. Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities linked to chromatin condensation failure.

    PubMed

    Boitrelle, F; Albert, M; Petit, J-M; Ferfouri, F; Wainer, R; Bergere, M; Bailly, M; Vialard, F; Selva, J

    2013-08-01

    Since an embryo's ability to grow to the blastocyst stage and implant can be improved by selection of a normal spermatozoon with a vacuole-free head, this study set out to determine the nature of small sperm vacuoles observed under high magnification (>×6300). For 15 infertile men with various sperm profiles, high-magnification microscopy was used to select motile, morphometrically normal spermatozoa with no vacuoles (n=450) or more than two small vacuoles (each of which occupied less than 4% of the head's area; n=450). Spermatozoa acrosome reaction status and degree of chromatin condensation were analysed. Three-dimensional deconvolution microscopy was used to accurately image the nucleus and acrosome at all depths in all spermatozoa. In all 450 spermatozoa with small vacuoles, the latter were seen to be abnormal, DNA-free nuclear concavities. Spermatozoa with small vacuoles were significantly more likely than vacuole-free spermatozoa to have noncondensed chromatin (39.8% versus 9.3%, respectively; P<0.0001). There was no significant difference between the two groups of spermatozoa in terms of acrosome reaction status. No association between chromatin condensation and acrosome reaction status was observed. Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities related to failure of chromatin condensation.

  15. Sperm shape abnormalities in carbaryl-exposed employees

    PubMed Central

    Wyrobek, A. J.; Watchmaker, G.; Gordon, L.; Wong, K.; Moore, D.; Whorton, D.

    1981-01-01

    Semen was collected from 50 men occupationally exposed to carbaryl (1-naphthyl methyl carbamate) in a produciton plant for durations of 1 to 18 years and compared to semen from a control group of 34 unexposed, newly-hired workers. Employment, fertility, health, personal data, and blood samples were collected for each individual. Semen samples were analyzed for changes in sperm count, morphology, and frequency of sperm carrying double flourescent bodies (YFF). As a group, the exposed workers showed a significantly higher proportion of sperm with abnormal head shapes than did the control group (p < 0.005). Age, smoking habits, and medical problems did not appear to affect this result. This finding appears to be limited to men working in the carbaryl production area at the time of sampling. Sperm count and YFF did not show similar differences, which may be because they are known to be statistically less sensitive to small changes. Formerly exposed workers (away from carbaryl for an average of 6.3 years) showed a marginally significant elevation in sperm abnormalities compared to controls (p < .05, one-tailed statistical analyses) suggesting that the increase in abnormal morphology may not be reversible. However, the question of reversibility is sensitive to confounding factors and small sample sizes and, therefore, requires further study. With these data a definitive link between carbaryl exposure and human seminal defects cannot be established. Although a distinct effect on sperm morphology was seen in the exposed group, the increases in sperm shape abnormalities were not related to exposure dose (estimated by number of years on the job or job classification during the year prior to semen collection). Inexplicably, the increases in sperm abnormalities were seen primarily in currently exposed men who had worked with carbaryl for less than approximately 6 years. These findings suggest the need for further study since other workplace-related factor(s) may be responsible

  16. Transcriptional activation of Xenopus class III genes in chromatin isolated from sperm and somatic nuclei.

    PubMed Central

    Wolffe, A P

    1989-01-01

    Xenopus sperm chromatin lacks class III transcription complexes and somatic histone H1. Inactive class III genes in sperm chromatin are easily programmed with transcription complexes de novo and transcribed in Xenopus oocyte nuclear extract. In contrast, repressed class III genes in somatic chromatin are not transcribed in the oocyte nuclear extract. Class III genes that are initially inactive or repressed in both types of chromatin can be efficiently transcribed in a cell free preparation of Xenopus eggs. Chromatin mediated repression of class III genes in somatic nuclei is reversible in Xenopus egg extract, but not in the oocyte nuclear extract. Any inhibition of transcription attributed to chromatin assembly onto a gene, will therefore depend on the extract in which transcription is assayed. Images PMID:2915929

  17. Colloidal centrifugation with Androcoll-E prolongs stallion sperm motility, viability and chromatin integrity.

    PubMed

    Johannisson, A; Morrell, J M; Thorén, J; Jönsson, M; Dalin, A-M; Rodriguez-Martinez, H

    2009-11-01

    The objective was to investigate the changes in stallion sperm quality (sperm motility, viability, membrane integrity and chromatin integrity) occurring during cool storage, and to study the effect of sperm selection by single layer colloidal centrifugation on these parameters of sperm quality. Spermatozoa from 3 stallions (10 ejaculates, 3-4 per stallion) were selected by centrifugation through a single layer of colloid (SLC). The resulting sperm preparations and the control samples (extended but unselected semen samples) were stored at 5 degrees C for 48h. Assessments of sperm quality, such as sperm motility, viability (SYBR-14/PI staining), membrane stability (Annexin-V/PI staining) and chromatin integrity, were performed on aliquots of the selected sperm preparations and unselected samples on the day of collection (3h) and after 24 and 48h of storage. In the SLC-selected sperm samples, sperm motility, sperm viability, proportions of spermatozoa with normal morphology and with intact chromatin were significantly better than in unselected samples (motility: 77+/-4% vs. 64+/-8% at 3h; P<0.001; viability: 79.5+/-9% vs. 64.7+/-9%, P<0.001; normal morphology 89+/-6% vs. 69+/-9%; chromatin integrity DFI 11.3+/-5% vs. 22.1+/-10%). Membrane stability, however, was not different in the SLC-selected and unselected samples (74.6+/-8% vs. 69.3+/-8%). The deterioration seen in sperm quality in the unselected samples was prevented by SLC, so that sperm viability, membrane stability and chromatin integrity were unchanged in the selected samples by 48h compared to 3h (P<0.001), whereas the unselected samples were significantly worse by 48h (P<0.001). Furthermore, it should be possible to send an aliquot of a normal insemination dose (i.e. unselected spermatozoa) overnight to a reference laboratory for analysis of both plasma membrane and chromatin integrity. In conclusion, centrifugation of stallion spermatozoa through a single layer of colloid is a useful technique for

  18. Evaluation of human sperm chromatin status after selection using a modified Diff-Quik stain indicates embryo quality and pregnancy outcomes following in vitro fertilization.

    PubMed

    Tavares, R S; Silva, A F; Lourenço, B; Almeida-Santos, T; Sousa, A P; Ramalho-Santos, J

    2013-11-01

    Sperm chromatin/DNA damage can be measured by a variety of assays. However, it has been reported that these tests may lose prognostic value in Assisted Reproductive Technology (ART) cycles when assessed in post-prepared samples, possibly due to the normalizing effect promoted by sperm preparation procedures. We have recently implemented a modified version of the Diff-Quik staining assay that allows for the evaluation of human sperm chromatin status in native samples, together with standard sperm morphology assessment. However, the value of this parameter in terms of predicting in vitro fertilization (IVF) and Intracytoplasmic sperm injection (ICSI) outcomes after sperm selection is unknown. In this study, data from 138 couples undergoing in vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) treatments showed that sperm chromatin integrity was significantly improved after density gradient centrifugation and swim up (p < 0.001), but no correlations were found with fertilization or embryo development rates (p > 0.05). However, sperm samples presenting lower percentages of damaged chromatin were associated with better quality (Grade I) embryos in both ART procedures (p < 0.05) and clinical pregnancy among IVF couples (p < 0.05). Furthermore, regression analysis confirmed the clinical value of Diff-Quik staining in predicting IVF (but not ICSI) clinical pregnancy (OR: 0.927, 95% CI: 0.871-0.985, p = 0.015), and a threshold value of 34.25% for this parameter was established. The proportion of IVF couples achieving a clinical pregnancy was reduced 1.9-fold when the percentage of abnormal dark staining was ≥34.25% (p = 0.05). In conclusion, the Diff-Quik staining assay provides useful information regarding ART success, particularly in IVF cycles, where some degree of 'natural' sperm selection may occur; but not in ICSI, where sperm selection is operator dependent. This quick and low-cost assay is suggested as an alternative method to detect

  19. Relationship of seminal reactive nitrogen and oxygen species and total antioxidant capacity with sperm DNA fragmentation in infertile couples with normal and abnormal sperm parameters.

    PubMed

    Khosravi, F; Valojerdi, M R; Amanlou, M; Karimian, L; Abolhassani, F

    2014-02-01

    The objective of this study was to investigate the association between the amount of superoxide anion, peroxynitrite as oxidative stress (OS) markers and total antioxidant capacity (TAC) with sperm DNA fragmentation in infertile men with abnormal semen parameters. Semen samples were obtained from 102 infertile couples and divided into groups with normal and abnormal semen parameters according to the World Health Organization (WHO). Peroxynitrite and superoxide anions were detected using spectrofluorometric assays combined with 2,7 dicholorofluorescein (DCF)-DA and 4-chloro-7-nitrobenzo-2-oxa -1, 3-diazole (NBD-CL). Colorimetric assay was used for evaluation of TAC, while DNA fragmentation was studied by using sperm chromatin dispersion test. Superoxide anion, peroxynitrite and DNA fragmentation were significantly higher in infertile couples with abnormal semen parameters as compared to infertile couples with normal semen (P < 0.01). TAC was significantly lower in infertile men with abnormal semen parameters (P < 0.01). There was also a significant positive correlation between OS markers with sperm DNA fragmentation (r = 0.59, P < 0.01 and r = 0.67, P < 0.01, respectively). We have found that imbalance between superoxide anion and peroxynitrite with antioxidant capacity in infertile men with abnormal sperm parameters is associated with higher sperm DNA fragmentation.

  20. Protection of cisplatin-induced spermatotoxicity, DNA damage and chromatin abnormality by selenium nano-particles.

    PubMed

    Rezvanfar, Mohammad Amin; Rezvanfar, Mohammad Ali; Shahverdi, Ahmad Reza; Ahmadi, Abbas; Baeeri, Maryam; Mohammadirad, Azadeh; Abdollahi, Mohammad

    2013-02-01

    Cisplatin (CIS), an anticancer alkylating agent, induces DNA adducts and effectively cross links the DNA strands and so affects spermatozoa as a male reproductive toxicant. The present study investigated the cellular/biochemical mechanisms underlying possible protective effect of selenium nano-particles (Nano-Se) as an established strong antioxidant with more bioavailability and less toxicity, on reproductive toxicity of CIS by assessment of sperm characteristics, sperm DNA integrity, chromatin quality and spermatogenic disorders. To determine the role of oxidative stress (OS) in the pathogenesis of CIS gonadotoxicity, the level of lipid peroxidation (LPO), antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and peroxynitrite (ONOO) as a marker of nitrosative stress (NS) and testosterone (T) concentration as a biomarker of testicular function were measured in the blood and testes. Thirty-two male Wistar rats were equally divided into four groups. A single IP dose of CIS (7 mg/kg) and protective dose of Nano-Se (2 mg/kg/day) were administered alone or in combination. The CIS-exposed rats showed a significant increase in testicular and serum LPO and ONOO level, along with a significant decrease in enzymatic antioxidants levels, diminished serum T concentration and abnormal histologic findings with impaired sperm quality associated with increased DNA damage and decreased chromatin quality. Coadministration of Nano-Se significantly improved the serum T, sperm quality, and spermatogenesis and reduced CIS-induced free radical toxic stress and spermatic DNA damage. In conclusion, the current study demonstrated that Nano-Se may be useful to prevent CIS-induced gonadotoxicity through its antioxidant potential.

  1. Increased aneuploidy rate in sperm with fragmented DNA as determined by the sperm chromatin dispersion (SCD) test and FISH analysis.

    PubMed

    Muriel, Lourdes; Goyanes, Vicente; Segrelles, Enrique; Gosálvez, Jaime; Alvarez, Juan G; Fernández, José Luis

    2007-01-01

    Previous studies suggest that sperm DNA fragmentation may be associated with aneuploidy. However, currently available tests have not made it possible to simultaneously perform DNA fragmentation and chromosomal analyses on the same sperm cell. The recently introduced sperm chromatin dispersion (SCD) test allows users to determine this relationship. Semen samples from 16 males, including 4 fertile donors, 7 normozoospermic, 3 teratozoospermic, 1 asthenozoospermic, and 1 oligoasthenoteratozoospermic, were processed for DNA fragmentation analysis by the SCD test using the Halosperm kit. Three-color fluorescence in situ hybridization (FISH) was performed on SCD-processed slides to determine aneuploidy for chromosomes X, Y, and 18. Spermatozoa with DNA fragmentation showed a 4.4 +/- 1.9-fold increase in diploidy rate and a 5.9 +/- 3.5-fold increase in disomy rate compared to spermatozoa without DNA fragmentation. The overall aneuploidy rate was 4.6 +/- 2.0-fold higher in sperm with fragmented DNA (Wilcoxon rank test: P < .001 in the 3 comparisons). A higher frequency of DNA fragmentation was found in sperm cells containing sex chromosome aneuploidies originated in both first and second meiotic divisions. The observed increase in aneuploidy rate in sperm with fragmented DNA may suggest that the occurrence of aneuploidy during sperm maturation may lead to sperm DNA fragmentation as part of a genomic screening mechanism developed to genetically inactivate sperm with a defective genomic makeup.

  2. Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.

    PubMed

    Burruel, Victoria; Klooster, Katie; Barker, Christopher M; Pera, Renee Reijo; Meyers, Stuart

    2014-10-13

    Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (<1 hr) P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.

  3. Diazinon alters sperm chromatin structure in mice by phosphorylating nuclear protamines

    SciTech Connect

    Pina-Guzman, B.; Solis-Heredia, M.J.; Quintanilla-Vega, B. . E-mail: mquintan@mail.cinvestav.mx

    2005-01-15

    Organophosphorus (OP) pesticides, widely used in agriculture and pest control, are associated with male reproductive effects, including sperm chromatin alterations, but the mechanisms underlying these effects are unknown. The main toxic action of OP is related to phosphorylation of proteins. Chemical alterations in sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis, contribute to male reproductive toxicity. Therefore, in the present study, we tested the ability of diazinon (DZN), an OP compound, to alter sperm chromatin by phosphorylating nuclear protamines. Mice were injected with a single dose of DZN (8.12 mg/kg, i.p.), and killed 8 and 15 days after treatment. Quality of sperm from epididymis and vas deferens was evaluated through standard methods and chromatin condensation by flow cytometry (DNA Fragmented Index parameters: DFI and DFI%) and fluorescence microscopy using chromomycin-A{sub 3} (CMA{sub 3}). Increases in DFI (15%), DFI% (4.5-fold), and CMA{sub 3} (2-fold) were observed only at 8 days post-treatment, indicating an alteration in sperm chromatin condensation and DNA damage during late spermatid differentiation. In addition, an increase of phosphorous content (approximately 50%) in protamines, especially in the phosphoserine content (approximately 73%), was found at 8 days post-treatment. Sperm viability, motility, and morphology showed significant alterations at this time. These data strongly suggest that spermatozoa exposed during the late steps of maturation were the targets of DZN exposure. The correlation observed between the phosphorous content in nuclear protamines with DFI%, DFI, and CMA{sub 3} provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin.

  4. New insights into protamine-like component organization in Mytilus galloprovincialis' sperm chromatin.

    PubMed

    Vassalli, Quirino Attilio; Caccavale, Filomena; Avagnano, Stefano; Murolo, Alessandra; Guerriero, Giulia; Fucci, Laura; Ausió, Juan; Piscopo, Marina

    2015-03-01

    We have analyzed Mytilus galloprovincialis' sperm chromatin, which consists of three protamine-like proteins, PL-II, PL-III, and PL-IV, in addition to a residual amount of the four core histones. We have probed the structure of this sperm chromatin through digestion with micrococcal nuclease (MNase) in combination with salt fractionation. Furthermore, we used the electrophoretic mobility shift assay to define DNA-binding mode of PL-II and PL-III and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. Although in literature it is reported that M. galloprovincialis' sperm chromatin lacks nucleosomal organization, our results obtained by MNase digestion suggest the existence of a likely unusual organization, in which there would be a more accessible location of PL-II/PL-IV when compared with PL-III and core histones. So, we hypothesize that in M. galloprovincialis' sperm chromatin organization DNA is wrapped around a PL-III protein core and core histones and PL-II and PL-IV are bound to the flanking DNA regions (similarly to somatic histone H1). Furthermore, we propose that PL's K/R ratio affects their DNA-binding mode and self-association ability as reported previously for somatic and sperm H1 histones.

  5. Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF.

    PubMed

    Niu, Zhi-Hong; Shi, Hui-Juan; Zhang, Hui-Qin; Zhang, Ai-Jun; Sun, Yi-Juan; Feng, Yun

    2011-11-01

    The aim of this study was to investigate whether the sperm chromatin structure assay (SCSA) results after swim-up are related to fertilization rates, embryo quality and pregnancy rates following in vitro fertilization (IVF). A total of 223 couples undergoing IVF in our hospital from October 2008 to September 2009 were included in this study. Data on the IVF process and sperm chromatin structure assay results were collected. Fertilization rate, embryo quality and IVF success rates of different DNA fragmentation index (DFI) subgroups and high DNA stainability (HDS) subgroups were compared. There were no significant differences in fertilization rate, clinical pregnancy or delivery rates between the DFI and HDS subgroups. However, the group with abnormal DFI had a lower good embryo rate. So, we concluded that the SCSA variables, either DFI or HDS after swim-up preparation, were not valuable in predicting fertilization failure or pregnancy rate, but an abnormal DFI meant a lower good embryo rate following IVF.

  6. Sperm DNA quality evaluated by comet assay and sperm chromatin structure assay in stallions after unilateral orchiectomy.

    PubMed

    Serafini, R; Varner, D D; Bissett, W; Blanchard, T L; Teague, S R; Love, C C

    2015-09-15

    Unilateral orchiectomy (UO) may interfere with thermoregulation of the remaining testis caused by inflammation surrounding the incision site, thus altering normal spermatogenesis and consequently sperm quality. Two measures of sperm DNA quality (neutral comet assay and the sperm chromatin structure assay [SCSA]) were compared before UO (0 days) and at 14, 30, and 60 days after UO to determine whether sperm DNA changed after a mild testis stress (i.e., UO). The percent DNA in the comet tail was higher at 14 and 60 days compared to 0 days (P < 0.05) after UO. All other comet tail measures (i.e., length, moment, migration) were higher at all time periods after UO compared to 0 days (P < 0.05). Two SCSA measures (mean-αt, mode-αt) increased at 14 days after UO (P < 0.05), whereas two measures (SD-αt and COMP-αt) did not change. This study identified a decrease in sperm DNA quality using both the neutral comet assay and the SCSA, which was not identified using traditional measures of sperm quality.

  7. Low lead environmental exposure alters semen quality and sperm chromatin condensation in northern Mexico.

    PubMed

    Hernández-Ochoa, Isabel; García-Vargas, Gonzalo; López-Carrillo, Lizbeth; Rubio-Andrade, Marisela; Morán-Martínez, Javier; Cebrián, Mariano E; Quintanilla-Vega, Betzabet

    2005-01-01

    We evaluated environmental-lead (Pb) effects on semen quality and sperm chromatin, considering Pb in seminal fluid (PbSF), spermatozoa (PbSpz), and blood (PbB) as exposure biomarkers in urban men (9.3 microg/dL PbB). Several individuals (44%) showed decreases in sperm quality; sperm concentration, motility, morphology and viability associated negatively with PbSpz, whereas semen volume associated negatively with PbSF. Multiple linear regression estimated PbSF and PbSpz thresholds for alterations in semen quality. Forty-eight percent of samples showed high values of nuclear chromatin condensation (NCD) positively associated with PbSF and zinc in spermatozoa (ZnSpz). ZnSpz values were higher than in fertile men. These results suggest that Pb may affect sperm chromatin by altering sperm Zn availability. PbB was not associated with semen quality or NCD, suggesting that Pb in semen compartments assesses better the amount of Pb in the reproductive tract; therefore, these are better biomarkers to evaluate toxicity at low Pb-exposure levels.

  8. Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure

    PubMed Central

    Kost, Nils; Kaiser, Sophie; Ostwal, Yogesh; Riedel, Dietmar; Stützer, Alexandra; Nikolov, Miroslav; Rathke, Christina; Renkawitz-Pohl, Renate; Fischle, Wolfgang

    2015-01-01

    Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure. PMID:25735749

  9. Presence and release of bovine sperm histone H1 during chromatin decondensation by heparin-glutathione.

    PubMed

    Sánchez-Vázquez, María Luisa; Flores-Alonso, Juan Carlos; Merchant-Larios, Horacio; Reyes, Rosalina

    2008-01-01

    During spermatogenesis, changes in sperm nuclear morphology are associated with the replacement of core somatic histones by protamines. Although protamines are the major nucleoproteins of mature sperm, not all species totally replace the histones. Histone H1, along with protamines, mediates chromatin condensation into an insoluble complex that is transcriptionally inactive. In vitro, heparin-reduced glutathione causes sperm decondensation, and the structures formed are morphologically similar to the in vivo male pronucleus. To study the participation of histone H1 in bovine sperm chromatin remodelling, we measured the presence and release of histone H1 by immunofluorescence, acetic acid-urea-triton-polyacrylamide gel electrophoresis, and immunoblotting. Nuclear decondensation was induced by 80 microM heparin and 15.0 mM reduced glutathione (GSH) for 7, 14, and 21 h at 37 degrees C. Additionally, nucleons, composed of nuclei isolated from the sperm, were decondensed with 20.0 microM heparin and 5.0 mM GSH for 4.0 h at 37 degrees C. Controls were incubated in buffer for similar periods of time. Immunofluorescent localization of histone H1 was carried out with mouse monoclonal antibody, and DNA localization was visualized by 0.001% quinacrine staining. Chromatin decondensation was accompanied by increased sperm nuclei and nucleon surface area. We observed that histone H1 was localized exclusively in the nuclei of intact sperm and nucleons. Histone H1 immunofluorescent intensity did not change in control samples but decreased over time in samples treated with heparin-GSH. There was a negative correlation between the surface area of sperm nuclei and immunohistochemical intensity of histone H1 (P < 0.05). Nucleon decondensation showed a similar relationship. By electrophoresis and immunoblotting, we verified the loss of histone H1 from the sperm and nucleons and its release into the incubation media. Based on these results, we propose that histone H1 is present in the

  10. Nuclease Footprints in Sperm Project Past and Future Chromatin Regulatory Events

    PubMed Central

    Johnson, Graham D.; Jodar, Meritxell; Pique-Regi, Roger; Krawetz, Stephen A.

    2016-01-01

    Nuclear remodeling to a condensed state is a hallmark of spermatogenesis. This is achieved by replacement of histones with protamines. Regions retaining nucleosomes may be of functional significance. To determine their potential roles, sperm from wild type and transgenic mice harboring a single copy insert of the human protamine cluster were subjected to Micrococcal Nuclease-seq. CENTIPEDE, a hierarchical Bayesian model, was used to identify multiple spatial patterns, "footprints", of MNase-seq reads along the sperm genome. Regions predicted by CENTIPEDE analysis to be bound by a regulatory factor in sperm were correlated with genomic landmarks and higher order chromatin structure datasets to identify potential roles for these factors in regulating either prior or post spermatogenic, i.e., early embryonic events. This approach linked robust endogenous protamine transcription and transgene suppression to its chromatin environment within topologically associated domains. Of the candidate enhancer-bound regulatory proteins, Ctcf, was associated with chromatin domain boundaries in testes and embryonic stem cells. The continuity of Ctcf binding through the murine germline may permit rapid reconstitution of chromatin organization following fertilization. This likely reflects its preparation for early zygotic genome activation and comparatively accelerated preimplantation embryonic development program observed in mouse as compared to human and bull. PMID:27184706

  11. Genes for embryo development are packaged in blocks of multivalent chromatin in zebrafish sperm.

    PubMed

    Wu, Shan-Fu; Zhang, Haiying; Cairns, Bradley R

    2011-04-01

    In mature human sperm, genes of importance for embryo development (i.e., transcription factors) lack DNA methylation and bear nucleosomes with distinctive histone modifications, suggesting the specialized packaging of these developmental genes in the germline. Here, we explored the tractable zebrafish model and found conceptual conservation as well as several new features. Biochemical and mass spectrometric approaches reveal the zebrafish sperm genome packaged in nucleosomes and histone variants (and not protamine), and we find linker histones high and H4K16ac absent, key factors that may contribute to genome condensation. We examined several activating (H3K4me2/3, H3K14ac, H2AFV) and repressing (H3K27me3, H3K36me3, H3K9me3, hypoacetylation) modifications/compositions genome-wide and find developmental genes packaged in large blocks of chromatin with coincident activating and repressing marks and DNA hypomethylation, revealing complex "multivalent" chromatin. Notably, genes that acquire DNA methylation in the soma (muscle) are enriched in transcription factors for alternative cell fates. Remarkably, whereas H3K36me3 is located in the 3' coding region of heavily transcribed genes in somatic cells, H3K36me3 is present in the promoters of "silent" developmental regulators in sperm, suggesting different rules for H3K36me3 in the germline and soma. We also reveal the chromatin patterns of transposons, rDNA, and tDNAs. Finally, high levels of H3K4me3 and H3K14ac in sperm are correlated with genes activated in embryos prior to the mid-blastula transition (MBT), whereas multivalent genes are correlated with activation at or after MBT. Taken together, gene sets with particular functions in the embryo are packaged by distinctive types of complex and often atypical chromatin in sperm.

  12. Sperm chromatin structure assay results in Nigerian men with unexplained infertility

    PubMed Central

    Kolade, Charles Oluwabukunmi

    2015-01-01

    Objective Several publications have established a relationship between sperm DNA damage and male factor infertility, based on data from America, Europe, and Asia. This study aimed to compare the extent of sperm DNA damage in sperm samples from Nigerian men with unexplained infertility and in sperm samples from a fertile group composed of sperm donors who had successfully impregnated a female partner naturally or through assisted conception. Methods A total of 404 men underwent male fertility evaluation at Androcare Laboratories and Cryobank participated in this study. Semen analysis and a sperm chromatin structure assay (SCSA) were performed on all subjects. Results The men in the unexplained infertility group were slightly older than the men in the fertile sperm group (36±10 years vs. 32±6 years, p=0.051). No significant difference was observed between the two groups in semen analysis parameters (p≥0.05). Men in the unexplained infertility group with normal semen parameters had a significantly higher DNA fragmentation index (DFI) than men in the fertile sperm group (27.5%±7.0% vs. 14.1%±5.3%, p<0.05). In the unexplained infertility group, 63% of the men had a DFI greater than 20%, compared to 4% in the fertile sperm group. In the unexplained infertility group, 15.2% of the subjects had a DFI greater than 30%, compared to 1% in the fertile sperm group. Conclusion Our study showed that the SCSA may be a more reliable predictor of fertility potential than traditional semen analysis in cases of unexplained infertility. PMID:26473109

  13. Sperm FISH and chromatin integrity in spermatozoa from a t(6;10;11) carrier.

    PubMed

    Olszewska, Marta; Huleyuk, Nataliya; Fraczek, Monika; Zastavna, Danuta; Wiland, Ewa; Kurpisz, Maciej

    2014-05-01

    Complex chromosome rearrangements (CCRs) are structurally balanced or unbalanced aberrations involving more than two breakpoints on two or more chromosomes. CCRs can be a potential reason for genomic imbalance in gametes, which leads to a drastic reduction in fertility. In this study, the meiotic segregation pattern, aneuploidy of seven chromosomes uninvolved in the CCR and chromatin integrity were analysed in the ejaculated spermatozoa of a 46,XY,t(6;10;11)(q25.1;q24.3;q23.1)mat carrier with asthenozoospermia and a lack of conception. The frequency of genetically unbalanced spermatozoa was 78.8% with a prevalence of 4:2 segregants of 38.2%, while the prevalence of the adjacent 3:3 mode was 35.3%. Analysis of the aneuploidy of chromosomes 13, 15, 18, 21, 22, X and Y revealed an approximately fivefold increased level in comparison with that of the control group, indicating the presence of an interchromosomal effect. Sperm chromatin integrity status was evaluated using chromomycin A3 and aniline blue staining (deprotamination), acridine orange test and TUNEL assay (sperm DNA fragmentation). No differences were found when comparisons were made with a control group. We suggest that the accumulation of genetically unbalanced spermatozoa, significantly increased sperm aneuploidy level and decreased sperm motility (20%, progressive) were not responsible for the observed lack of reproductive success in the analysed infertile t(6;10;11) carrier. Interestingly, in the case described herein, a high level of sperm chromosomal imbalance appears not to be linked to sperm chromatin integrity status.

  14. A model for the importance of zinc in the dynamics of human sperm chromatin stabilization after ejaculation in relation to sperm DNA vulnerability.

    PubMed

    Björndahl, Lars; Kvist, Ulrik

    2011-02-01

    The focus of this review is the dual functions of the sperm chromatin stabilization and how external factors can interfere with these functions. Zinc depletion after ejaculation allows for rapid and total sperm chromatin decondensation without addition of exogenous disulfide cleaving agents. Zinc depletion without concomitant repulsion of chromatin fibers induces another type of stability that requires exogenous disulfide cleaving agents to allow decondensation. It is essential to extend the present concept, that the sperm chromatin stability is based on disulfide bridges only, to include also the functions of Zn(2+). It is suggested that the chromatin stability of the ejaculated human spermatozoon is rapidly reversible due to the dual function of Zn(2+) that stabilizes the structure and prevents the formation of excess disulfide bridges by a single mechanism: the formation of zinc bridges involving protamine thiols of cysteine and potentially also imidazole groups of histidine. Extraction of zinc from the freshly ejaculated spermatozoon allows two totally different biological results: (1) immediate decondensation if chromatin fibers concomitantly are induced to repel (e.g., through phosphorylation in the ooplasm) and (2) thiols freed from Zn(2+) are available to form disulfide bridges creating a superstabilized chromatin. Spermatozoa in the zinc rich prostatic fluid (in first ejaculated fraction) represent physiology. Extraction of chromatin zinc can be caused by unphysiological exposure of spermatozoa to the zinc chelating and oxidative seminal vesicular fluid, a situation common to most assisted reproductive techniques (ART) laboratories where the entire ejaculate is collected into a single container in which spermatozoa and secretions are mixed during at least 30 min. Some men in infertile couples have low content of sperm chromatin zinc due to loss of zinc during ejaculation and liquefaction. Tests for sperm DNA integrity may give false negative results due to

  15. Impact of sperm genome decay on Day-3 embryo chromosomal abnormalities from advanced-maternal-age patients.

    PubMed

    Kaarouch, Ismail; Bouamoud, Nouzha; Louanjli, Noureddine; Madkour, Aicha; Copin, Henri; Benkhalifa, Moncef; Sefrioui, Omar

    2015-10-01

    Infertile male patients often exhibit unconventional semen parameters, including DNA fragmentation, chromatin dispersion, and aneuploidy-collectively referred to as sperm genome decay (SGD). We investigated the correlation of SGD to embryo chromosomal abnormalities and its effect on clinical pregnancy rates in patients with advanced maternal age (AMA) (>40 years) who were undergoing intracytoplasmic sperm injection-preimplantation genetic screening (ICSI-PGS). Three groups were assessed: patients with AMA and male partners with normal sperm (AMA-N); AMA patients and male partners presenting with SGD (AMA-SGD); and young fertile female patients and male partners with SGD (Y-SGD). We found a significant increase in embryonic chromosomal abnormalities-polyploidy, nullisomy, mosaicism, and chaotic anomaly rates-when semen parameters are altered (76% vs. 67% and 66% in AMA-SGD vs. AMA-N and Y-SGD groups, respectively). Statistical analysis showed a correlation between SGD and aneuploidies of embryonic chromosomes 13, 16, 21, X, and Y, as well as negative clinical outcomes. Incorporation of molecular sperm analyses should therefore significantly minimize the risk of transmission of chromosomal anomalies from spermatozoa to embryos, and may provide better predictors of pregnancy than conventional sperm analyses. We also demonstrated that an ICSI-PGS program should be implemented for SGD patients in order to limit transmission of chromosomal paternal anomalies and to improve clinical outcome.

  16. Sperm Chromatin Immaturity Observed in Short Abstinence Ejaculates Affects DNA Integrity and Longevity In Vitro

    PubMed Central

    Salian, Sujith Raj; Kumar, Dayanidhi; Singh, Vikram Jeet; D’Souza, Fiona; Kalthur, Guruprasad; Kamath, Asha; Adiga, Satish Kumar

    2016-01-01

    Background The influence of ejaculatory abstinence (EA) on semen parameters and subsequent reproductive outcome is still debatable; hence understanding the impact of EA on sperm structural and functional integrity may provide a valuable information on predicting successful clinical outcome. Objective To understand the influence of EA on sperm chromatin maturity, integrity, longevity and global methylation status. Methods This experimental prospective study included 76 ejaculates from 19 healthy volunteers who provided ejaculates after observing 1, 3, 5 and 7 days of abstinence. Sperm chromatin maturity, DNA integrity and global methylation status were assessed in the neat ejaculate. Sperm motility, DNA integrity and longevity were assessed in the processed fraction of the fresh and frozen-thawed ejaculates to determine their association with the length of EA. Results Spermatozoa from 1 day ejaculatory abstinence (EA-1) displayed significantly higher level of sperm chromatin immaturity in comparison to EA-3 (P < 0.05) and EA-5 (P < 0.01) whereas; the number of 5-methyl cytosine immunostained spermatozoa did not vary significantly across groups. On the other hand, in vitro incubation of processed ejaculate from EA-1 resulted in approximately 20 and 40 fold increase in the DNA fragmented spermatozoa at the end of 6 and 24h respectively (P < 0.01–0.001). Conclusion Use of short-term EA for therapeutic fertilization would be a clinically valuable strategy to improve the DNA quality. However, use of such spermatozoa after prolonged incubation in vitro should be avoided as it can carry a substantial risk of transmitting DNA fragmentation to the oocytes. PMID:27043437

  17. [Ultrastructural observation of morphologically abnormal sperm: Advances in studies and application].

    PubMed

    Wang, Jia-xiong; Shi, Yi-chao; Yang, Shen-min

    2016-01-01

    Sperm ultrastructural abnormalities are often associated with sperm motility, the integrity of genetic material, and the fertilization potential. The investigation of sperm ultrastructural abnormalities is based on the evolution of microscopy techniques. In his paper, we review the improvement of the microscopy techniques and the ultrastructure of several specific morphological defects and he apoptotic spermatogenic cells in order to expound the significance of sperm ultrastructural observation in clinical practice. We deem it necessary to analyze the sperm ultrastructure before exploring the pathology and adopting assisted reproductive technology for some special patients with teratozoospermia.

  18. Dialkyl Phosphate Urinary Metabolites and Chromosomal Abnormalities in Human Sperm

    PubMed Central

    Figueroa, Zaida I.; Young, Heather A.; Meeker, John D.; Martenies, Sheena E.; Barr, Dana Boyd; Gray, George; Perry, Melissa J.

    2015-01-01

    Background The past decade has seen numerous human health studies seeking to characterize the impacts of environmental exposures, such as organophosphate (OP) insecticides, on male reproduction. Despite an extensive literature on OP toxicology, many hormone-mediated effects on the testes are not well understood. Objectives This study investigated environmental exposures to OPs and their association with the frequency of sperm chromosomal abnormalities (i.e., disomy) among adult men. Methods Men (n=159) from a study assessing the impact of environmental exposures on male reproductive health were included in this investigation. Multi-probe fluorescence in situ hybridization (FISH) for chromosomes X, Y, and 18 was used to determine XX18, YY18, XY18 and total disomy in sperm nuclei. Urine was analyzed using gas chromatography coupled with mass spectrometry for concentrations of dialkyl phosphate (DAP) metabolites of OPs [dimethylphosphate (DMP); dimethylthiophosphate (DMTP); dimethyldithiophosphate (DMDTP); diethylphosphate (DEP); diethylthiophosphate (DETP); and diethyldithiophosphate (DEDTP)]. Poisson regression was used to model the association between OP exposures and disomy measures. Incidence rate ratios (IRRs) were calculated for each disomy type by exposure quartiles for most metabolites, controlling for age, race, BMI, smoking, specific gravity, total sperm concentration, motility, and morphology. Results A significant positive trend was seen for increasing IRRs by exposure quartiles of DMTP, DMDTP, DEP and DETP in XX18, YY18, XY18 and total disomy. A significant inverse association was observed between DMP and total disomy. Findings for total sum of DAP metabolites concealed individual associations as those results differed from the patterns observed for each individual metabolite. Dose-response relationships appeared nonmonotonic, with most of the increase in disomy rates occurring between the second and third exposure quartiles and without additional

  19. Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)

    USGS Publications Warehouse

    Jenkins, Jill A.; Draugelis-Dale, Rassa O.; Pinkney, Alfred E.; Iwanowicz, Luke R.; Blazer, Vicki

    2015-01-01

    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

  20. Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens).

    PubMed

    Jenkins, J A; Draugelis-Dale, R O; Pinkney, A E; Iwanowicz, L R; Blazer, V S

    2015-03-15

    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

  1. Development of a novel flow cytometric approach to evaluate fish sperm chromatin using fixed samples

    USGS Publications Warehouse

    Jenkins, Jill A.

    2013-01-01

    The integrity of the paternal DNA is essential for the accurate transmission of genetic information, yet fertilization is not inhibited by chromatin breakage. Some methods are available for the sensitive detection of DNA damage and can be applied in studies of environmental toxicology, carcinogenesis, aging, and assisted reproduction techniques in both clinical and experimental settings. Because semen samples obtained from remote locations undergo chromatin damage prior to laboratory assessment, the present study was undertaken to evaluate treatments for effective chromatin staining in the development of a DNA fragmentation assay using fixed milt from yellow perch (Perca flavescens). Similar to the sperm chromatin structure assay (SCSA), susceptibility of nuclear DNA to acid-induced denaturation was measured by flow cytometry (FCM). Use of 10% buffered formalin for milt fixation allowed easier peak discrimination than 4% paraformaldehyde. The effects of time and temperature of incubation in 0.08 N HCl were evaluated in order to determine the ideal conditions for promoting DNA decondensation and making strand breaks more available for staining and detection by FCM. The best results were obtained with incubation at 37°C for 1 minute, followed by cold propidium iodide staining for 30 minutes.

  2. The effect of heracleum persicum (Golpar) oil and alcoholic extracts on sperm parameters and chromatin quality in mice

    PubMed Central

    Taghizabet, Neda; Mangoli, Esmat; Anbari, Fatemeh; Masoodi, Seyed Ali; Talebi, Ali Reza; Mazrooei, Malihe

    2016-01-01

    Background: Evaluating the significance and the effects of plant-derived drugs on laboratory animal’s fertility was recognized. There was antioxidant activity reported from Heracleum persicum (Golpar). Objective: Current study aims to study the antioxidant effect of Golpar extracts on sperm parameters and chromatin quality in mice. Materials and Methods: Eighteen adult male mice were divided to 3 groups (10 wk old, 35 gr weight): group1 received hydro alcoholic extract (1000 mg/kg, ip), group 2 received oil extract (200 mg/kg, ip) and group 3 serving as the sham control group that received sterile water. Finally, left cauda epididymis of each animal was dissected and sperm analysis was done accordingly. To asses sperm chromatin and DNA quality, we used aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3) and acridine orange (AO) staining. Results: Progressive and non-progressive sperm motility were significantly increased in group 1 in comparison with group 3 (p=0.032). There was an increasing trend in progressive sperm motility and decreasing trend in non-progressive sperm motility in group 2 in comparison with group 3, but the differences were not significant (p=0.221 and p=0.144, respectively). According to the sperm chromatin quality, the results of TB and AO tests revealed significant differences (p=0.004, p=0.000, respectively) between those groups and showed that the extracts of Golpar cause DNA damage, but no differences can be observed between them in AB and CMA3 staining (p>0.05). Conclusion: The results showed that Heracleum persicum extracts may improve sperm motility. Also, it has harmful effects on sperm chromatin condensation and DNA integrity in mice. PMID:27525319

  3. Scrotal insulation and its relationship to abnormal morphology, chromatin protamination and nuclear shape of spermatozoa in Holstein-Friesian and Belgian Blue bulls.

    PubMed

    Rahman, Mohammad Bozlur; Vandaele, Leen; Rijsselaere, Tom; Maes, Dominiek; Hoogewijs, Maarten; Frijters, Adrie; Noordman, Jakomien; Granados, Ana; Dernelle, Eric; Shamsuddin, Mohammed; Parrish, John J; Van Soom, Ann

    2011-10-15

    The objectives of this study were to identify the stages of spermatogenesis susceptible to elevated testicular temperature in terms of sperm motility, viability, morphology, chromatin protamination and nuclear shape. The latter two valuable parameters are not included in routine semen analysis. Scrotal insulation (SI) was applied for 48 h in 2 Holstein-Friesian (HF) and 2 Belgian Blue (BB) bulls and semen was collected at 7 d intervals along with semen collection of a non-insulated bull of each breed. Semen samples were frozen and assigned to 4 groups: period 1 (preinsulation) = -7 d and 0 d, where 0 d = initiation of SI after semen collection; period 2 = 7 d (sperm presumed in the epididymis during SI); period 3 = 14 d to 42 d (cells presumed at spermiogenesis and meiosis stages during SI); period 4 = 49 d to 63 d (cells presumed at spermatocytogenesis stage during SI). The percentages of progressively motile and viable spermatozoa as assessed by computer-assisted sperm analysis (CASA) and fluorescence microscopy, respectively were decreased whereas abnormal sperm heads, nuclear vacuoles and tail defects were increased at period 3 (P < 0.05) compared to period 1, 2 or 4 in SI bulls of both HF and BB breeds. Protamine deficient spermatozoa as observed by chromomycin A(3) (CMA(3)) staining were more present (P < 0.05) at period 2 and 3 in both breeds compared to period 1 or 4. Sperm nuclear shape as determined by Fourier harmonic amplitude (FHA) was most affected by heat stress during period 3 (P < 0.01) and a higher response was observed in BB bulls than HF bulls. In conclusion, sperm cells at the spermiogenic and meiotic stages of development are more susceptible to heat stress. The lack of chromatin protamination is the most pertinent result of heat stress, together with subtle changes in sperm head shape, which can be detected by FHA but not by conventional semen analysis.

  4. Comparative sperm chromatin structure assay measurements on epiillumination and orthogonal axes flow cytometers

    SciTech Connect

    Evenson, D.; Jost, L.; Gandour, D.; Gandour, D.; Rhodes, L.

    1995-04-01

    The sperm chromatin structure assay (SCSA) measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ, and was developed on two Ortho flow cytometers, an FC200 and a cytofluorograf 30 (BDIS), both having orthogonal axes of fluorochrome excitation, emission, and sample flow. Sperm cells are first treated with a pH 1.4 buffer to denature DNA in situ and then stained with the metachromatic dye acridine orange (AO). The metachromatic fluorescence measured reflects relative amounts of denatured (red fluorescence) and native (green fluorescence) DNA present per cell. The extent of DNA denaturation is quantified by the calculated parameter alpha t [{alpha}{sub t} = red/(red + green) fluorescence]. Alpha t variables important for correlations with fertility and toxicant-induced chromatin damage include mean (X{alpha}{sub t}), standard deviation (SD{alpha}{sub t}), and cells outside the main population (COMP{alpha}{sub t}). This study showed that the SCSA can be successfully run on two epiillumination-type instruments, an Ortho ICP22A and Skatron Argus {trademark}, and two additional orthogonal axes instruments, a Becton Dickinson FACScan {trademark} and a Coulter Elite {trademark}. Epiillumination instruments produced a different fluorescence distribution than orthogonal instruments, but the resulting {alpha}{sub t} values showed strong conformity and interpretation of results was the same. SCSA values obtained on the Coultier Elite {trademark} were most similar to the Cytofluorograf 30; the FACScan {trademark} green fluorescence distribution was narrower and allowed resolution of cell doublets. Neither orthogonal instrument has the ability to directly calculate {alpha}{sub t} values. Listmode data from these instruments were transferred to an off-line personal computer (PC) for calculation of {alpha}{sub t} values using LIST-VIEW {trademark} software. 28 refs., 5 figs., 2 tabs.

  5. Protective role of RAD50 on chromatin bridges during abnormal cytokinesis.

    PubMed

    Schröder-Heurich, Bianca; Wieland, Britta; Lavin, Martin F; Schindler, Detlev; Dörk, Thilo

    2014-03-01

    Faithful chromosome segregation is required for preserving genomic integrity. Failure of this process may entail chromatin bridges preventing normal cytokinesis. To test whether RAD50, a protein normally involved in DNA double-strand break repair, is involved in abnormal cytokinesis and formation of chromatin bridges, we used immunocytochemical and protein interaction assays. RAD50 localizes to chromatin bridges during aberrant cytokinesis and subsequent stages of the cell cycle, either decorating the entire bridge or focally accumulating at the midbody zone. Ionizing radiation led to an ∼4-fold increase in the rate of chromatin bridges in an ataxia telangiectatica mutated (ATM)-dependent manner in human RAD50-proficient fibroblasts but not in RAD50-deficient cells. Cells with a RAD50-positive chromatin bridge were able to continue cell cycling and to progress through S phase (44%), whereas RAD50 knockdown caused a deficiency in chromatin bridges as well as an ∼4-fold prolonged duration of mitosis. RAD50 colocalized and directly interacted with Aurora B kinase and phospho-histone H3, and Aurora B kinase inhibition led to a deficiency in RAD50-positive bridges. Based on these observations, we propose that RAD50 is a crucial factor for the stabilization and shielding of chromatin bridges. Our study provides evidence for a hitherto unknown role of RAD50 in abnormal cytokinesis.

  6. Influence of sperm fertilising concentration, sperm selection method and sperm capacitation procedure on the incidence of numerical chromosomal abnormalities in IVF early bovine embryos.

    PubMed

    Demyda-Peyrás, Sebastián; Dorado, Jesús; Hidalgo, Manuel; Moreno-Millán, Miguel

    2015-01-01

    The occurrence of numerical chromosomal aberrations, widely described as a major cause of mortality in in vitro-produced (IVP) embryos, has been linked to several factors. In the present study we investigated the effect of sperm fertilising concentration and semen handling (sperm selection and capacitation) before IVF on the rate of numerical chromosomal abnormalities in bovine embryos. In all, 466 IVP cattle embryos were karyotyped throughout three sequential experiments, analysing the effects of sperm fertilising concentration (0.1, 1.0 or 10×10(6) spermatozoa mL(-1)), selection method (unselected or Percoll-selected spermatozoa) and capacitation medium (bovine serum albumin (BSA), heparin or their combination). The percentage of normal (diploid) and aberrant (haploid, polyploid or aneuploid) embryos was noted in each experiment. The rate of numerical chromosomal abnormalities was mainly affected by sperm fertilising concentration (P<0.01) and, to a lesser extent, by the sperm capacitation medium (P<0.05). Polyploidy and haploidy rates were only affected by sperm fertilising concentration (P<0.05). Interestingly, the sperm selection technique used in the present study did not reduce the incidence of chromosome abnormalities in IVP cattle embryos (P>0.05). Finally, aneuploidy rates were not affected during the experiments (P>0.05), which suggests that they are not related to sperm-related factors. On the basis of these results, we conclude that sperm fertilising concentration is the 'paternal' key factor that affects the rate of numerical chromosomal abnormalities in IVP bovine embryos. By making small adjustments to fertilising protocols, the rate of cytogenetically aberrant embryos can be markedly reduced.

  7. Sedimentation properties in density gradients correspond with levels of sperm DNA fragmentation, chromatin compaction and binding affinity to hyaluronic acid.

    PubMed

    Torabi, Forough; Binduraihem, Adel; Miller, David

    2017-03-01

    Mature spermatozoa bind hyaluronic acid in the extracellular matrix via hyaladherins. Immature spermatozoa may be unable to interact because they do not express the appropriate hyaladherins on their surface. Fresh human semen samples were fractionated using differential density gradient centrifugation (DDGC) and the ability of these fractions to bind hyaluronic acid was evaluated. The presence of sperm hyaladherins was also assessed. CD44 was located mainly on the acrosome and equatorial segment and became more restricted to the equatorial segment in capacitated spermatozoa. Hyaluronic acid-TRITC (hyaluronic acid conjugated with tetramethylrhodamine isothiocyanante), a generic hyaluronic-acid-binding reagent, labelled the membrane and the neck region, particularly after capacitation. Sperm populations obtained after DDGC or after interaction with hyaluronic acid were assessed for DNA fragmentation and chromatin maturity. Strong relationships between both measures and sperm sedimentation and hyaluronic-acid-binding profiles were revealed. Capacitation enhanced hyaluronic acid binding of both DDGC-pelleted sperm and sperm washed free of seminal fluid. In conclusion, hyaladherins were detected on human sperm and a higher capacity for sperm hyaluronic-acid-binding was shown to correspond with their DDGC sedimentation profiles and with lower levels of DNA fragmentation and better chromatin maturity. Capacitation induced changes in the distribution and presence of hyaladherins may enhance hyaluronic-acid-binding.

  8. Sperm ultrastructure, morphometry, and abnormal morphology in American black bears (Ursus americanus).

    PubMed

    Brito, L F C; Sertich, P L; Stull, G B; Rives, W; Knobbe, M

    2010-11-01

    The objective of this study was to describe sperm ultrastructure, morphometry, and abnormal morphology in American black bears. Electroejaculation was successful in 53.8% (7/13) of the attempts, but urine contamination was common. Epididymal sperm samples were also obtained from five bears. Sperm had a paddle-like head shape and the ultrastructure was similar to that of most other mammals. The most striking particularity of black bear sperm ultrastructure was a tightening of the nucleus in the equatorial region. Although the differences were not significant in all bears, the overall decrease in sperm nucleus dimensions during transport from the caput epididymis to the cauda suggested increasing compaction of the nucleus during maturation. For ejaculated sperm, nucleus length, width, and base width were 4.9, 3.7, and 1.8 μm, respectively, whereas sperm head length, width, and base width were 6.6, 4.8, and 2.3 μm, and midpiece, tail (including midpiece), and total sperm lengths were 9.8, 68.8, and 75.3 μm. Evaluation of sperm cytoplasmic droplets in the epididymis revealed that proximal droplets start migrating toward a distal position in the caput epididymis and that the process was mostly completed by the time sperm reached the cauda epididymis. The proportion of morphologically normal sperm in the ejaculate was 35.6%; the most prevalent sperm defects were distal cytoplasmic droplets and bent/coiled tails. The morphology of abnormal sperm and the underlying ultrastructural defects were similar to that in other large domestic animals thus suggesting similar underlying pathogenesis of specific sperm defects and similar effects on fertility.

  9. Correlation between male age, WHO sperm parameters, DNA fragmentation, chromatin packaging and outcome in assisted reproduction technology.

    PubMed

    Nijs, M; De Jonge, C; Cox, A; Janssen, M; Bosmans, E; Ombelet, W

    2011-06-01

    In the human, male ageing results in reproductive hormonal and cellular changes that can influence semen quality (volume, motility, concentration and morphology) and ultimately result in a reduced fertilising capacity and a longer 'time to pregnancy' for ageing men as well as an increased risk for miscarriage. This prospective cohort study of 278 patients undergoing a first in vitro fertilisation or intracytoplasmic sperm injection treatment was undertaken to examine whether patient's age was reflected in sperm motility, concentration, morphology as well as in DNA fragmentation (DFI) and immature chromatin (unprocessed nuclear proteins and/or poorly condensed chromatin) as measured by the sperm chromatin structure assay. This study also investigated the possible influence of male age (after correcting for female age) on their fertilising capacity, on obtaining a pregnancy and a healthy baby at home. Logistic regression analysis did not reveal any male age-related influences on sperm parameters like concentration, motility or morphology. No significant male age-related increase in DFI or immature chromatin was demonstrable for these patients. Elevated male age, after correcting for female age, was not related to lower fertilisation rates or significant decreases in the chance for a healthy baby at home.

  10. Accurate sperm morphology assessment predicts sperm function.

    PubMed

    Abu Hassan Abu, D; Franken, D R; Hoffman, B; Henkel, R

    2012-05-01

    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.

  11. The Drosophila chromosomal protein Mst77F is processed to generate an essential component of mature sperm chromatin

    PubMed Central

    2016-01-01

    In most animals, the bulk of sperm DNA is packaged with sperm nuclear basic proteins (SNBPs), a diverse group of highly basic chromosomal proteins notably comprising mammalian protamines. The replacement of histones with SNBPs during spermiogenesis allows sperm DNA to reach an extreme level of compaction, but little is known about how SNBPs actually function in vivo. Mst77F is a Drosophila SNBP with unique DNA condensation properties in vitro, but its role during spermiogenesis remains unclear. Here, we show that Mst77F is required for the compaction of sperm DNA and the production of mature sperm, through its cooperation with protamine-like proteins Mst35Ba/b. We demonstrate that Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus. The cleavage of Mst77F is very similar to the processing of protamine P2 during human spermiogenesis and notably leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links. Despite the rapid evolution of SNBPs, sperm chromatin condensation thus involves remarkably convergent mechanisms in distantly related animals. PMID:27810970

  12. Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function.

    PubMed

    Johnston, S D; Satake, N; Zee, Y; López-Fernández, C; Holt, W V; Gosálvez, J

    2012-06-01

    This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic

  13. Assessment of chromosomal abnormalities in sperm of infertile men using sperm karyotyping and multicolour fluorescence in situ hybridization (FISH)

    SciTech Connect

    Moosani, N.; Martin, R.H.

    1994-09-01

    Individuals with male factor infertility resulting from idiopathic oligo-, astheno- or teratozoospermia are frequently offered IVF in an attempt to increase their chances of having a child. A concern remains whether these infertile males have an elevated risk of transmitting chromosomal abnormalities to their offspring. Sperm chromosomal complements from these men were assayed using the human sperm/hamster oocyte fusion system and fluorescence in situ hybridization (FISH) on sperm nuclei. For each of 5 infertile patients, 100 sperm karyotypes were analyzed and multicolour FISH analysis was performed on a minimum of 10,000 sperm nuclei for each chromosome-specific DNA probe for chromosomes 1 (pUC1.77), 12 (D12Z3), X (XC) and Y (DYZ3). As a group, the infertile patients showed increased frequencies of both numerical ({chi}{sup 2}=17.26, {proportional_to} <0.001) and total abnormalities ({chi}{sup 2}=7.78, {proportional_to} <0.01) relative to control donors when assessed by sperm karyotypes. Analysis of sperm nuclei by FISH indicated a significant increase in the frequency of disomy for chromosome 1 in three of the five patients as compared to control donors ({chi}{sup 2}>8.35, {proportional_to} <0.005). In addition, the frequency of XY disomy was significantly higher in four of the five patients studied by FISH ({chi}{sup 2}>10.58, {proportional_to}<0.005), suggesting that mis-segregation caused by the failure of the XY bivalent to pair may play a role in idiopathic male infertility.

  14. Sperm chromatin integrity in young men with no experiences of infertility and men from idiopathic infertility couples.

    PubMed

    Rybar, R; Markova, P; Veznik, Z; Faldikova, L; Kunetkova, M; Zajicova, A; Kopecka, V; Rubes, J

    2009-06-01

    Damage to the genetic component of spermatozoa seems to play the main role in a majority of cases where current approaches fail to reveal the specific cause of male infertility. In this study, we compared semen quality in men assigned to two defined groups: men from couples with unexplained infertility - idiopathic infertility (A) and young men with no experiences of infertility (B). All samples were examined by standard ejaculate analysis and sperm chromatin structure assay (SCSA). Sperm chromatin damage was significantly higher in men from group A than in those from group B. Similar results were obtained by comparison of men from group A (all men were normozoospermic) with normozoospermic men from group B. According to these results, we can suppose that chromatin disorders may be the causal factor of subfertility or infertility in some of these men. No evidence for a strong association between chromatin disorders and standard parameters of ejaculates was found. We failed to confirm a relationship between smoking and sperm quality in men from any of the investigated groups. SCSA is a method that facilitates the identification of infertile men who otherwise show normal semen variables.

  15. Zinc-silicon interactions influencing sperm chromatin integrity and testicular cell development in the rat as measured by flow cytometry.

    PubMed

    Evenson, D P; Emerick, R J; Jost, L K; Kayongo-Male, H; Stewart, S R

    1993-04-01

    Flow-cytometric procedures were used to determine effects of dietary Zn and Si variations on rat testicular cell development, including integrity of caudal epididymal sperm chromatin structure defined as the susceptibility of DNA to denaturation in situ. Concentrations of 4 (deficient), 12 (adequate), and 500 (excessive) mg of Zn/kg of diet were used with Si concentrations of 0 (low), 540 (medium), and 2,700 (high) mg/kg of diet in a 3 x 3 factorial arrangement. Three-week-old Sprague-Dawley male rats were fed the experimental diets for 8 wk. Rats fed the Zn-deficient/Si-low diet demonstrated significant deviations in the ratio of testicular cell types present, including a reduction of S phase and total haploid cells. Furthermore, approximately 50% of epididymal sperm had a significant decrease in resistance to DNA denaturation in situ. In the Zn-deficient/Si-medium treatment, the effects of Si on animal and testicular growth, distribution of testicular cell types, and sperm chromatin structure integrity were quite similar to the effects of the Zn-adequate diets. A toxic effect of Zn on sperm chromatin structure integrity observed in the Zn-excess/Si-medium treatment seemed to be counteracted by Si in the Zn-excess/Si-high treatment. Silicon at medium and high levels seems to affect Zn metabolism through potentiation and antagonistic reactions, respectively. Zinc deficiency likely disrupts the normal sperm chromatin quaternary structure in which Zn plays a role by providing stability and resistance to DNA denaturation in situ.

  16. DNA integrity of canine spermatozoa during chill storage assessed by the sperm chromatin dispersion test using bright-field or fluorescence microscopy.

    PubMed

    Hidalgo, M; Urbano, M; Ortiz, I; Demyda-Peyras, S; Murabito, M R; Gálvez, M J; Dorado, J

    2015-08-01

    The objective of this study was to evaluate the effect of chill storage on canine sperm DNA fragmentation assessed by the sperm chromatin dispersion test using bright-field microscopy with Wright solution (sDF-B) or fluorescence microscopy with propidium iodide (sDF-F). The relationship and agreement between the results obtained with both staining methods were analyzed. The values of DNA fragmentation indexes (sDF-F and sDF-B) were compared at each time of chill storage (0, 24, 48, 72, and 96 hours). Additionally, the sperm DNA fragmentation rate (slope) was compared between the methods during chill storage. Good agreement and no significant differences between values obtained with both staining procedures were observed. Finally, the effect of chill storage for up to 96 hours was assessed on sperm motility parameters and DNA fragmentation indexes. Significant differences were found after 48 hours of chill storage, obtaining greater values of fragmented DNA. Progressive sperm motility was lower just after 96 hours of chill storage, and no effect was found in total sperm motility. In conclusion, the Sperm-Halomax kit, developed for canine semen and based on the sperm chromatin dispersion test, can be used accurately under bright-field or fluorescence microscopy to assess the sperm DNA integrity of canine semen during chill storage. The sperm DNA fragmentation index increased after 48 hours of chill storage, thereby detecting sperm damage earlier than other routine sperm parameters, such as sperm motility.

  17. The Usefulness of Selected Physicochemical Indices, Cell Membrane Integrity and Sperm Chromatin Structure in Assessments of Boar Semen Sensitivity

    PubMed Central

    Wysokińska, A.; Kondracki, S.; Iwanina, M.

    2015-01-01

    The present work describes experiments undertaken to evaluate the usefulness of selected physicochemical indices of semen, cell membrane integrity and sperm chromatin structure for the assessment of boar semen sensitivity to processes connected with pre-insemination procedures. The experiments were carried out on 30 boars: including 15 regarded as providers of sensitive semen and 15 regarded as providers of semen that is little sensitive to laboratory processing. The selection of boars for both groups was based on sperm morphology analyses, assuming secondary morphological change incidence in spermatozoa as the criterion. Two ejaculates were manually collected from each boar at an interval of 3 to 4 months. The following analyses were carried out for each ejaculate: sperm motility assessment, sperm pH measurement, sperm morphology assessment, sperm chromatin structure evaluation and cell membrane integrity assessment. The analyses were performed three times. Semen storage did not cause an increase in the incidence of secondary morphological changes in the group of boars considered to provide sperm of low sensitivity. On the other hand, with continued storage there was a marked increase in the incidence of spermatozoa with secondary morphological changes in the group of boars regarded as producing more sensitive semen. Ejaculates of group I boars evaluated directly after collection had an approximately 6% smaller share of spermatozoa with undamaged cell membranes than the ejaculates of boars in group II (p≤0.05). In the process of time the percentage of spermatozoa with undamaged cell membranes decreased. The sperm of group I boars was characterised with a lower sperm motility than the semen of group II boars. After 1 hour of storing diluted semen, the sperm motility of boars producing highly sensitive semen was already 4% lower (p≤0.05), and after 24 hours of storage it was 6.33% lower than that of the boars that produced semen with a low sensitivity. Factors

  18. Methamphetamine induces abnormal sperm morphology, low sperm concentration and apoptosis in the testis of male rats.

    PubMed

    Nudmamud-Thanoi, S; Thanoi, S

    2011-08-01

    Methamphetamine has been reported to be an important drug in the field of reproductive toxicology. The aim of this study was to investigate the effects of methamphetamine administrations on sperm morphology, sperm concentration and apoptotic activity inside seminiferous tubule in male rats. Rats were administered a dose of 8 mg kg(-1) , intraperitoneally (IP), for acute group and a dose of 4 mg kg(-1) , IP, once daily for 14 days for sub-acute group. Percentage of normal sperm morphology was decreased in acute group when compared with control. Total numbers of sperm count were significantly decreased in acute and sub-acute groups. Apoptotic activities were most abundant in the seminiferous tubules of acute treated animals with a highly significant increase in the number of apoptotic cells per tubule. Those effects of methamphetamine seem to be dose-dependent. The results suggest that methamphetamine not only works as drug of abuse in central nervous system, but also in gametogenesis of males.

  19. Abnormal meiotic recombination in infertile men and its association with sperm aneuploidy.

    PubMed

    Ferguson, Kyle A; Wong, Edgar Chan; Chow, Victor; Nigro, Mark; Ma, Sai

    2007-12-01

    Defects in early meiotic events are thought to play a critical role in male infertility; however, little is known regarding the relationship between early meiotic events and the chromosomal constitution of human sperm. Thus, we analyzed testicular tissue from 26 men (9 fertile and 17 infertile men), using immunofluorescent techniques to examine meiotic chromosomes, and fluorescent in situ hybridization to assess sperm aneuploidy. Based on a relatively small sample size, we observed that 42% (5/12) of men with impaired spermatogenesis displayed reduced genome-wide recombination when compared to the fertile men. Analysis of individual chromosomes showed chromosome-specific defects in recombination: chromosome 13 and 18 bivalents with only a single crossover and chromosome 21 bivalents lacking a crossover were more frequent among the infertile men. We identified two infertile men who displayed a novel meiotic defect in which the sex chromosomes failed to recombine: one man had an absence of sperm in the testes, while the other displayed increased sex chromosome aneuploidy in the sperm, resulting in a 45,X abortus after intracytoplasmic sperm injection. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. Recombination between the sex chromosomes may be a useful indicator for identifying men at risk of producing chromosomally abnormal sperm. An understanding of the molecular mechanisms that contribute to sperm aneuploidy in infertile men could aid in risk assessment for couples undergoing assisted reproduction.

  20. Unusual chromatin structural organization in the sperm head of a murid rodent from southern Africa: the red veld rat, Aethomys chrysophilus type B.

    PubMed

    Breed, W G

    1997-11-01

    The structural organization of the chromatin of cauda epididymal spermatozoa of the red veld rat Aethomys chrysophilus type B was investigated by fluorescence microscopy after staining with DNA specific dyes and by transmission electron microscopy after incubation with Triton X100, dithiothreitol, and SDS. Staining with DNA dyes showed variation in intensity of fluorescence of the sperm chromatin, with an anterior spherical region staining far more intensely than the surrounding chromatin. Transmission electron microscopy of these spermatozoa indicated that this region was composed of cords and fibres. This chromatin region dispersed more readily than the surrounding chromatin when spermatozoa were incubated with the detergents, and it is suggested that, unlike the rest of the sperm chromatin, it may be a histone-rich region, with protamine(s) being either scarce or absent.

  1. Polychlorinated biphenyls and dibenzofurans increased abnormal sperm morphology without alterations in aneuploidy: The Yucheng study.

    PubMed

    Hsu, Ping-Chi; Li, Ming-Chieh; Lee, Yeu-Chin; Kuo, Pao-Lin; Guo, Yueliang Leon

    2016-12-01

    In 1979, more than 2000 persons ingested rice oil contaminated with polychlorinated biphenyls and polychlorinated dibenzofurans; this event was called the "Yucheng accident." An increased percentage of oligospermia, reduced ability of sperm to penetrate oocytes, and reduced percentage of male offspring were reported in Yucheng men. This study examined whether the sperm sex ratio and chromosome aneuploidy are responsible for our observed findings in Yucheng men. In 1999-2000, Yucheng men and their neighborhood referents aged 37-50 years were recruited for physical examination, followed by semen analysis. The semen samples were analyzed for chromosomal aneuploidy through fluorescent in situ hybridization according to an established procedure in our laboratory. A total of 50 Yucheng men and 34 neighborhood referents volunteered to participate in the study. Although abnormal morphology was mildly increased, no differences were observed in sperm percentages, with normal numbers of chromosomes X, Y, and 8 in the two groups. The percentage of sperm with aneuploidy of the sex chromosomes or chromosome 8 and of that with diploidy did not vary between both groups. The normal X/Y sperm ratio was not different between the groups. However, among Yucheng men, 8% had a normal X/Y sperm ratio of >1.4, and no neighborhood referent showed such an elevated X/Y ratio. Chromosomal aneuploidy was not elevated in Yucheng men. The mechanisms underlying the reduced sperm capability of oocyte penetration and changed offspring sex ratio in Yucheng men remain undetermined.

  2. Implication of sperm chromosomal abnormalities in recurrent abortion and multiple implantation failure.

    PubMed

    Caseiro, Ana Lara; Regalo, Ana; Pereira, Elisa; Esteves, Telma; Fernandes, Fernando; Carvalho, Joaquim

    2015-10-01

    Currently, some infertility treatment centres provide sperm karyotype analysis, although the impact of sperm chromosomal abnormalities on fertility is not yet fully understood. Several studies using fluorescence in-situ hybridization (FISH) to analyse sperm chromosomal constitution discovered that the incidence of aneuploidy is increased in individuals with a history of repeated abortion or implantation failure and is even higher in cases of oligoasthenoteratozoospermia (OAT), abnormal somatic karyotype or in spermatozoa retrieved directly from the testis or epididymis, showing that the application of FISH in these cases may be of some benefit for improving the reproductive outcome. This article presents the results of clinical trials of FISH analysis on spermatozoa, the medical indications for performing this examination, its results in infertile patients and the advantages when performing genetic counselling prior to treatment. Also discussed is the possibility of applying the latest techniques of genetic analysis in these cases and the potential benefits for improving the prognosis of male infertility.

  3. X-y interactions underlie sperm head abnormality in hybrid male house mice.

    PubMed

    Campbell, Polly; Nachman, Michael W

    2014-04-01

    The genetic basis of hybrid male sterility in house mice is complex, highly polygenic, and strongly X linked. Previous work suggested that there might be interactions between the Mus musculus musculus X and the M. m. domesticus Y with a large negative effect on sperm head morphology in hybrid males with an F1 autosomal background. To test this, we introgressed the M. m. domesticus Y onto a M. m. musculus background and measured the change in sperm morphology, testis weight, and sperm count across early backcross generations and in 11th generation backcross males in which the opportunity for X-autosome incompatibilities is effectively eliminated. We found that abnormality in sperm morphology persists in M. m. domesticus Y introgression males, and that this phenotype is rescued by M. m. domesticus introgressions on the X chromosome. In contrast, the severe reductions in testis weight and sperm count that characterize F1 males were eliminated after one generation of backcrossing. These results indicate that X-Y incompatibilities contribute specifically to sperm morphology. In contrast, X-autosome incompatibilities contribute to low testis weight, low sperm count, and sperm morphology. Restoration of normal testis weight and sperm count in first generation backcross males suggests that a small number of complex incompatibilities between loci on the M. m. musculus X and the M. m. domesticus autosomes underlie F1 male sterility. Together, these results provide insight into the genetic architecture of F1 male sterility and help to explain genome-wide patterns of introgression across the house mouse hybrid zone.

  4. X–Y Interactions Underlie Sperm Head Abnormality in Hybrid Male House Mice

    PubMed Central

    Campbell, Polly; Nachman, Michael W.

    2014-01-01

    The genetic basis of hybrid male sterility in house mice is complex, highly polygenic, and strongly X linked. Previous work suggested that there might be interactions between the Mus musculus musculus X and the M. m. domesticus Y with a large negative effect on sperm head morphology in hybrid males with an F1 autosomal background. To test this, we introgressed the M. m. domesticus Y onto a M. m. musculus background and measured the change in sperm morphology, testis weight, and sperm count across early backcross generations and in 11th generation backcross males in which the opportunity for X–autosome incompatibilities is effectively eliminated. We found that abnormality in sperm morphology persists in M. m. domesticus Y introgression males, and that this phenotype is rescued by M. m. domesticus introgressions on the X chromosome. In contrast, the severe reductions in testis weight and sperm count that characterize F1 males were eliminated after one generation of backcrossing. These results indicate that X–Y incompatibilities contribute specifically to sperm morphology. In contrast, X–autosome incompatibilities contribute to low testis weight, low sperm count, and sperm morphology. Restoration of normal testis weight and sperm count in first generation backcross males suggests that a small number of complex incompatibilities between loci on the M. m. musculus X and the M. m. domesticus autosomes underlie F1 male sterility. Together, these results provide insight into the genetic architecture of F1 male sterility and help to explain genome-wide patterns of introgression across the house mouse hybrid zone. PMID:24504187

  5. Assessment of Chromatin Maturity in Human Spermatozoa: Useful Aniline Blue Assay for Routine Diagnosis of Male Infertility

    PubMed Central

    Chakroun, Nozha; Ben Zarrouk, Soumaya; Sellami, Hanen; Kebaili, Sahbi; Rebai, Tarek; Keskes, Leila

    2013-01-01

    During spermatogenesis, sperm chromatin undergoes structural changes and results in a high condensation. This nuclear compaction would be useful as a predictor of sperm fertilization capacity and pregnancy outcome. We purpose to evaluate firstly the relationship among chromatin maturity assessed by aniline blue staining (AB) and the semen parameters in infertile men. Secondly, we analyzed whether the sperm gradient density centrifugation is effective to select mature spermatozoa. Fifty-one ejaculates were investigated by semen analysis and stained for chromatin condensation with AB to distinguish between unstained mature sperm and stained immature sperm. AB was applied also on 12 ejaculates which proceeded by density gradient centrifugation to compare the rates of immature sperm before and after selection. Neat semen were divided into two groups: G1 (n = 31): immature sperm <20% and G2 (n = 20): immature sperm ≥20%. No significant differences were detected in sperm concentration, motility, and normal morphology between G1 and G2. However, the rates of some morphology abnormalities were higher in G2: head abnormalities (P = 0.01) and microcephalic sperm (P = 0.02). We founded significant correlation between sperm immaturity and acrosome abnormalities (r = 0.292; P = 0.03). Sperm selection has significantly reduced the rates of immature sperm. A better understanding of chromatin structure and its impact on the sperm potential is needed to explore male infertility. PMID:24198830

  6. Prevalence of chromosomal abnormalities and Y chromosome microdeletion among men with severe semen abnormalities and its correlation with successful sperm retrieval

    PubMed Central

    Mascarenhas, Mariano; Thomas, Sumi; Kamath, Mohan S.; Ramalingam, Ramya; Kongari, Ann Marie; Yuvarani, S; Srivastava, Vivi M.; George, Korula

    2016-01-01

    AIM: To estimate the prevalence of chromosomal abnormalities and Y chromosome microdeletion among men with azoospermia and severe oligozoospermia and its correlation with successful surgical sperm retrieval. SETTING AND DESIGN: A prospective study in a tertiary level infertility unit. MATERIALS AND METHODS: In a prospective observation study, men with azoospermia and severe oligozoospermia (concentration <5 million/ml) attending the infertility center underwent genetic screening. Peripheral blood karyotype was done by Giemsa banding. Y chromosome microdeletion study was performed by a multiplex polymerase chain reaction. RESULTS: The study group consisted of 220 men, 133 of whom had azoospermia and 87 had severe oligozoospermia. Overall, 21/220 (9.5%) men had chromosomal abnormalities and 13/220 (5.9%) men had Y chromosome microdeletions. Chromosomal abnormalities were seen in 14.3% (19/133) of azoospermic men and Y chromosome microdeletions in 8.3% (11/133). Of the 87 men with severe oligozoospermia, chromosomal abnormalities and Y chromosome microdeletions were each seen in 2.3% (2/87). Testicular sperm aspiration was done in 13 men and was successful in only one, who had a deletion of azoospermia factor c. CONCLUSIONS: Our study found a fairly high prevalence of genetic abnormality in men with severe semen abnormalities and a correlation of genetic abnormalities with surgical sperm retrieval outcomes. These findings support the need for genetic screening of these men prior to embarking on surgical sperm retrieval and assisted reproductive technology intracytoplasmic sperm injection. PMID:27803587

  7. Light microscopy morphological characteristics of the sperm flagellum may be related to axonemal abnormalities.

    PubMed

    Mitchell, V; Sigala, J; Ballot, C; Jumeau, F; Barbotin, A L; Duhamel, A; Rives, N; Rigot, J M; Escalier, D; Peers, M C

    2015-03-01

    Although electron microscopy provides a detailed analysis of ultrastructural abnormalities, this technique is not available in all laboratories. We sought to determine whether certain characteristics of the flagellum as assessed by light microscopy were related to axonemal abnormalities. Forty-one patients with an absence of outer dynein arms (type I), a lack of a central complex (type III) and an absence of peripheral doublets (type IV) were studied. Sperm morphology was scored according to David's modified classification. Flagella with an irregular thickness were classified as being of normal length, short or broken. There were correlations between missing outer dynein arms and abnormal, short or coiled flagellum. Type III patients showed the highest flagellar defects (a short (P = 0.0027) or an absent flagellum (P = 0.011)). Just over 68% of the irregular flagella were short in Type III patients, whereas this value was only 34.5% in type I and 26.4% in type IV (P = 0.002). There was a negative correlation between misassembly and spermatozoa of irregular flagella (r = -0.79; P = 0.019). It is concluded that light microscopy analysis of flagellum abnormalities may help provide a correct diagnosis, identify sperm abnormalities with fertility potentials and outcomes in assisted reproduction technologies and assess the genetic risk.

  8. Short-term storage of human spermatozoa in electrolyte-free medium without freezing maintains sperm chromatin integrity better than cryopreservation.

    PubMed

    Riel, Jonathan M; Yamauchi, Yasuhiro; Huang, Thomas T F; Grove, John; Ward, Monika A

    2011-09-01

    Previous attempts to maintain human spermatozoa without freezing were based on short-term storage in component-rich medium and led to fast decline in motility and increased incidence of chromosome breaks. Here we report a new method in which sperm are maintained without freezing in an electrolyte-free medium (EFM) composed of glucose and bovine serum albumin. Human sperm were stored in EFM or human tubal fluid medium (HTFM) or were cryopreserved, and their motility, viability, and DNA integrity were examined at different intervals. Cryopreservation led to significant decline in sperm motility and viability and induced DNA fragmentation. Sperm stored in EFM maintained motility and viability for up to 4 and 7 wk, respectively, much longer than sperm stored in HTFM (<2 and <4 wk, respectively). DNA integrity, assessed with comet assay, was also maintained significantly better in EFM than in HTFM. One-week storage in EFM yielded motility and viability similar to that of cryopreserved sperm, but DNA integrity was significantly higher, resembling that of fresh sperm. After several weeks of storage in EFM, sperm were able to activate oocytes, undergo chromatin remodeling, and form normal zygotic chromosomes after intracytoplasmic sperm injection. This study demonstrated that human spermatozoa can be stored in EFM without freezing for several weeks while maintaining motility, viability, and chromatin integrity and that 1-wk storage in EFM offers better protection of sperm DNA integrity than cryopreservation. Sperm storage in EFM may become a viable option for the physicians working in assisted reproduction technology clinics, which would avoid cryodamage.

  9. Galactosylceramidase deficiency causes sperm abnormalities in the mouse model of globoid cell leukodystrophy

    SciTech Connect

    Luddi, A.; Strazza, M.; Carbone, M.; Moretti, E.; Costantino-Ceccarini, E. . E-mail: costantino@unisi.it

    2005-03-10

    The classical recessive mouse mutant, 'the twitcher,' is one of the several animal models of the human globoid cell leukodystrophy (Krabbe disease) caused by a deficiency in the gene encoding the lysosomal enzyme galactosylceramidase (GALC). The failure to hydrolyze galactosylceramide (gal-cer) and galactosylsphingosine (psychosine) leads to degeneration of oligodendrocytes and severe demyelination. Substrate for GALC is also the galactosyl-alkyl-acyl-glycerol (GalAAG), precursor of the seminolipid, the most abundant glycolipid in spermatozoa of mammals. In this paper, we report the pathobiology of the testis and sperm in the twitcher mouse and demonstrate the importance of GALC for normal sperm maturation and function. The GALC deficit results in accumulation of GalAAG in the testis of the twitcher mouse. Morphological studies revealed that affected spermatozoa have abnormally swollen acrosomes and angulation of the flagellum mainly at midpiece-principal piece junction. Multiple folding of the principal piece was also observed. Electron microscopy analysis showed that in the twitcher sperm, acrosomal membrane is redundant, detached from the nucleus and folded over. Disorganization and abnormal arrangements of the axoneme components were also detected. These results provide in vivo evidence that GALC plays a critical role in spermiogenesis.

  10. Detection of structural and numerical chomosomal abnormalities by ACM-FISH analysis in sperm of oligozoospermic infertility patients

    SciTech Connect

    Schmid, T E; Brinkworth, M H; Hill, F; Sloter, E; Kamischke, A; Marchetti, F; Nieschlag, E; Wyrobek, A J

    2003-11-10

    Modern reproductive technologies are enabling the treatment of infertile men with severe disturbances of spermatogenesis. The possibility of elevated frequencies of genetically and chromosomally defective sperm has become an issue of concern with the increased usage of intracytoplasmic sperm injection (ICSI), which can enable men with severely impaired sperm production to father children. Several papers have been published about aneuploidy in oligozoospermic patients, but relatively little is known about chromosome structural aberrations in the sperm of these patients. We examined sperm from infertile, oligozoospermic individuals for structural and numerical chromosomal abnormalities using a multicolor ACM FISH assay that utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical chromosomal abnormalities plus two categories of structural aberrations: duplications and deletions of 1pter and 1cen, and chromosomal breaks within the 1cen-1q12 region. There was a significant increase in the average frequencies of sperm with duplications and deletions in the infertility patients compared with the healthy concurrent controls. There was also a significantly elevated level of breaks within the 1cen-1q12 region. There was no evidence for an increase in chromosome-1 disomy, or in diploidy. Our data reveal that oligozoospermia is associated with chromosomal structural abnormalities suggesting that, oligozoospermic men carry a higher burden of transmissible, chromosome damage. The findings raise the possibility of elevated levels of transmissible chromosomal defects following ICSI treatment.

  11. Deficiency of the Chromatin Regulator Brpf1 Causes Abnormal Brain Development*

    PubMed Central

    You, Linya; Zou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-01-01

    Epigenetic mechanisms are important in different neurological disorders, and one such mechanism is histone acetylation. The multivalent chromatin regulator BRPF1 (bromodomain- and plant homeodomain-linked (PHD) zinc finger-containing protein 1) recognizes different epigenetic marks and activates three histone acetyltransferases, so it is both a reader and a co-writer of the epigenetic language. The three histone acetyltransferases are MOZ, MORF, and HBO1, which are also known as lysine acetyltransferase 6A (KAT6A), KAT6B, and KAT7, respectively. The MORF gene is mutated in four neurodevelopmental disorders sharing the characteristic of intellectual disability and frequently displaying callosal agenesis. Here, we report that forebrain-specific inactivation of the mouse Brpf1 gene caused early postnatal lethality, neocortical abnormalities, and partial callosal agenesis. With respect to the control, the mutant forebrain contained fewer Tbr2-positive intermediate neuronal progenitors and displayed aberrant neurogenesis. Molecularly, Brpf1 loss led to decreased transcription of multiple genes, such as Robo3 and Otx1, important for neocortical development. Surprisingly, elevated expression of different Hox genes and various other transcription factors, such as Lhx4, Foxa1, Tbx5, and Twist1, was also observed. These results thus identify an important role of Brpf1 in regulating forebrain development and suggest that it acts as both an activator and a silencer of gene expression in vivo. PMID:25568313

  12. Deficiency of the chromatin regulator BRPF1 causes abnormal brain development.

    PubMed

    You, Linya; Zou, Jinfeng; Zhao, Hong; Bertos, Nicholas R; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-03-13

    Epigenetic mechanisms are important in different neurological disorders, and one such mechanism is histone acetylation. The multivalent chromatin regulator BRPF1 (bromodomain- and plant homeodomain-linked (PHD) zinc finger-containing protein 1) recognizes different epigenetic marks and activates three histone acetyltransferases, so it is both a reader and a co-writer of the epigenetic language. The three histone acetyltransferases are MOZ, MORF, and HBO1, which are also known as lysine acetyltransferase 6A (KAT6A), KAT6B, and KAT7, respectively. The MORF gene is mutated in four neurodevelopmental disorders sharing the characteristic of intellectual disability and frequently displaying callosal agenesis. Here, we report that forebrain-specific inactivation of the mouse Brpf1 gene caused early postnatal lethality, neocortical abnormalities, and partial callosal agenesis. With respect to the control, the mutant forebrain contained fewer Tbr2-positive intermediate neuronal progenitors and displayed aberrant neurogenesis. Molecularly, Brpf1 loss led to decreased transcription of multiple genes, such as Robo3 and Otx1, important for neocortical development. Surprisingly, elevated expression of different Hox genes and various other transcription factors, such as Lhx4, Foxa1, Tbx5, and Twist1, was also observed. These results thus identify an important role of Brpf1 in regulating forebrain development and suggest that it acts as both an activator and a silencer of gene expression in vivo.

  13. Epididymal hypo-osmolality induces abnormal sperm morphology and function in the estrogen receptor alpha knockout mouse.

    PubMed

    Joseph, Avenel; Shur, Barry D; Ko, CheMyong; Chambon, Pierre; Hess, Rex A

    2010-05-01

    Estrogen receptor-alpha (ESR1) is highly expressed in the efferent ductules of all species studied as well as in the epididymal epithelium in mice and other select species. Male mice lacking ESR1 (Esr1KO) are infertile, but transplantation studies demonstrated that Esr1KO germ cells are capable of fertilization when placed in a wild-type reproductive tract. These results suggest that extratesticular regions, such as the efferent ductules and epididymis, are the major source of pathological changes in Esr1KO males. Previous studies have shown alterations in ion and fluid transporters in the efferent duct and epididymal epithelia of Esr1KO males, leading to misregulation of luminal fluid pH. To determine the effect of an altered epididymal milieu on Esr1KO sperm, we assayed sperm morphology in the different regions of the epididymis. Sperm recovered from the epididymis exhibited abnormal flagellar coiling and increased incidence of spontaneous acrosome reactions, both of which are consistent with exposure to abnormal epididymal fluid. Analysis of the epididymal fluid revealed that the osmolality of the Esr1KO fluid was reduced relative to wild type, consistent with prior reports of inappropriate fluid absorption from the efferent ductules. This, along with the finding that morphological defects increased with transit through the epididymal duct, suggests that the anomalies in sperm are a consequence of the abnormal luminal environment. Consistent with this, incubating Esr1KO sperm in a more wild-type-like osmotic environment significantly rescued the abnormal flagellar coiling. This work demonstrates that Esr1KO mice exhibit an abnormal fluid environment in the lumen of the efferent ducts and epididymis, precluding normal sperm maturation and instead resulting in progressive deterioration of sperm that contributes to infertility.

  14. Changeability of sperm chromatin structure during liquid storage of ovine semen in milk-egg yolk- and soybean lecithin-based extenders and their relationships to field-fertility.

    PubMed

    Khalifa, Tarek; Lymberopoulos, Aristotelis

    2013-12-01

    The aim of this experiment was to study the effect of semen extender on sperm chromatin structure and to correlate chromatin integrity with field-fertility of preserved ram semen. Ejaculates of at least 2 × 10(9) sperm/ml and 70 % progressive motility were collected using an artificial vagina from Chios rams (n = 11, 4-6 years old), split-diluted to 1 × 10(9) sperm/ml with milk-egg yolk- and soybean lecithin (Ovixcell®)-based extenders, packaged in 0.5-ml straws and examined after 6, 24 and 48 h of storage at 5 ± 1 °C. Evaluation endpoints were computer-assisted sperm motion analysis, fluorescence-based analysis of chromatin structure by chromomycin A3 and acridine orange assays, and 65-day pregnancy rate (PR) of 34- to 36-h preserved semen after intra-cervical insemination of ewes (n = 154) in progestagen-synchronized estrus. Neither extender nor storage time had any influence on incidence of decondensed chromatin. Unlike Ovixcell® extender, deterioration of sperm motility (P < 0.01) and chromatin stability (P < 0.005) was detected after 48 h of storage in milk-egg yolk extender. Sperm motility accounted for 14.4-18.5 % of variations in chromatin integrity (P < 0.001). No significant difference was found in PR of Ovixcell®- and milk-egg yolk-stored semen. Nevertheless, PR differed between rams (14.3-71.4 %; P < 0.025). Chromatin integrity explained 10.2-56.3 % of variations in PR (P < 0.05-0.01). A pronounced decline in PR (19.1 %) was observed when percentages of decondensed and destabilized chromatin have reached thresholds of 10.5-30 % and 4-9 %, respectively. In conclusion, Ovixcell® is superior to milk-egg yolk extender in preserving chromatin stability and motility. Chromatin defects are negatively associated with sperm fertility.

  15. Abnormal sperm morphology in mouse germ cells after short-term exposures to acetamiprid, propineb, and their mixture.

    PubMed

    Rasgele, Pinar Göç

    2014-03-01

    Pesticides are one of the most potent environmental contaminants, which accumulate in biotic and abiotic components of ecosystems. Acetamiprid (Acm), a neonicotinoid insecticide, and Propineb (Pro), a dithiocarbamate fungicide, are widely used to control sucking insects and fungal infections on crops, respectively. The present study was undertaken to investigate the genotoxic effects of these compounds, individually and in mixtures, in mouse germ cells by using the sperm morphology assay. Mice were injected intraperitoneally with 0.625, 1.25, and 2.50 μg mL⁻¹ of Acm, 12.5, 25, and 50 μg mL⁻¹ of Pro, and their mixture at the same concentrations over 24 and 48 h. Acm did not significantly increase the percentage of abnormal sperm at any concentration. The frequency of abnormal sperm significantly increased after 24 and 48 h of exposure to 50 μg mL⁻¹ of Pro. The mixtures of 2.50 μg mL⁻¹ of Acm and 50 μg mL⁻¹ of Pro induced sperm abnormalities antagonistically both after 24 and 48 h of exposure. Results suggest that Acm was non-genotoxic for mouse germ cells, while Pro may have been a germ cell mutagen due to the observed increase in the frequency of sperm abnormalities. However, to gain better insight into the mutagenicity and DNA damaging potential of both of these pesticides, further studies at molecular level should be done.

  16. Ability of abnormally-shaped human spermatozoa to adhere to and penetrate zona-free hamster eggs: correlation with sperm morphology and postincubation motility.

    PubMed

    Bronson, Richard A; Bronson, Susan K; Oula, Lucila D

    2007-01-01

    A body of evidence indicates that morphologically abnormal human spermatozoa may exhibit impaired ability to fertilize. Yet teratospermia has widely varying etiologies, including associations with varicoceles, following fever, cigarette smoking, and exposure to polychlorinated biphenyls. Abnormalities of sperm shape in mice have also been shown to be associated with autosomal gene mutations. These varying causes of teratospermia could have different molecular consequences reflected in altered sperm function. We studied the ability of morphologically abnormal human sperm to penetrate zona-free hamster eggs as a measure of their ability to undergo an acrosome reaction and gamete membrane fusion. Motile sperm from ejaculates containing 15% normal sperm or less, as judged by World Health Organization (1999) criteria, were recovered by ISolate density centrifugation and capacitated by overnight incubation. Zona-free hamster eggs were inseminated with 1 x 10(6) motile capacitated cells and scored for sperm penetration after 3 hours of coincubation. A significant trend was found between the percent of abnormal spermatozoa within the ejaculate and impaired egg-penetrating ability, reflected in the percent of eggs penetrated, the number of penetrating sperm per egg, and the number of sperm adherent to the oolemma. Because only acrosome-reacted human spermatozoa adhere to the oolemma, these results support the notion that abnormally shaped sperm may exhibit an impaired ability to undergo an acrosome reaction. A correlation was also noted between the loss of motility of sperm following overnight incubation and impairment of their ability to undergo gamete membrane fusion. These results confirm prior findings at the level of the zona pellucida that abnormally shaped sperm exhibit functional abnormalities. However, a wide variation was observed between men in the behavior of such sperm, including occasionally high rates of egg penetration. These observations suggest that

  17. Studies on Brahma rasayana in male swiss albino mice: Chromosomal aberrations and sperm abnormalities

    PubMed Central

    Guruprasad, K. P.; Mascarenhas, Roshan; Gopinath, P. M.; Satyamoorthy, K.

    2010-01-01

    Ayurveda, the Indian holistic healthcare system encompasses traditional medicines with a principle of creating harmony and maintaining balance within the natural rhythms of the body. Rasayana is one of the branches of Ayurveda frequently used as rejuvenant therapy to overcome many discomforts and prevent diseases. It has been reported that rasayanas have immunomodulatory, antioxidant and antitumor functions. However, the genotoxic potential of many rasayanas remains to be evaluated. The present study was undertaken to assess the role of Brahma rasayana(BR) on genotoxicity in vivo in a mouse test system. The older mice (9 months) were orally fed with rasayana for 8 weeks. The treated groups showed no signs of dose-dependent toxicity at the dosage levels tested. The body weight loss/gain and feed consumption were unaffected at tested doses. Furthermore, sperm abnormalities and chromosomal aberrations were insignificant in the treatment group when compared to controls. However, there was a marginal increase in sperm count in the BR treated animals. These findings clearly indicate that there are no observed adverse genotoxic effects elicited by BR in experimental animals such as mice. PMID:21829300

  18. Studies on Brahma rasayana in male swiss albino mice: Chromosomal aberrations and sperm abnormalities.

    PubMed

    Guruprasad, K P; Mascarenhas, Roshan; Gopinath, P M; Satyamoorthy, K

    2010-01-01

    Ayurveda, the Indian holistic healthcare system encompasses traditional medicines with a principle of creating harmony and maintaining balance within the natural rhythms of the body. Rasayana is one of the branches of Ayurveda frequently used as rejuvenant therapy to overcome many discomforts and prevent diseases. It has been reported that rasayanas have immunomodulatory, antioxidant and antitumor functions. However, the genotoxic potential of many rasayanas remains to be evaluated. The present study was undertaken to assess the role of Brahma rasayana(BR) on genotoxicity in vivo in a mouse test system. The older mice (9 months) were orally fed with rasayana for 8 weeks. The treated groups showed no signs of dose-dependent toxicity at the dosage levels tested. The body weight loss/gain and feed consumption were unaffected at tested doses. Furthermore, sperm abnormalities and chromosomal aberrations were insignificant in the treatment group when compared to controls. However, there was a marginal increase in sperm count in the BR treated animals. These findings clearly indicate that there are no observed adverse genotoxic effects elicited by BR in experimental animals such as mice.

  19. The disruption in actin-perinuclear theca interactions are related with changes induced by cryopreservation observed on sperm chromatin nuclear decondensation of boar semen.

    PubMed

    Gutiérrez-Pérez, O; Juárez-Mosqueda, M L; Mota, D; Trujillo, M E

    2011-02-01

    The perinuclear theca (PT) is a cytoskeletal structure that surrounds the mammal sperm nucleus which must be disrupted once the sperm has penetrated the oocyte to permit normal chromatin decondensation and formation of male pronucleus. F-actin is a thermo sensitive protein found in the equatorial segment which is involved in the stability of PT. It has been reported that cryopreservation induces alterations in nuclear decondensation of spermatozoa, which have been interpreted as an over condensation. The aims of the present study were identified the presence of changes in sperm sPT integrity of frozen-thawed boar spermatozoa and its effect in sperm nuclei decondensation; and whether changes in the actin cytoskeleton are involved using an in vitro model to test probably differences in a chemical decondensation (DTT/heparin) between fresh (FS) and frozen-thawed (TS) spermatozoa. Results showed an increase on sPT damage in TS (P<0.001), and significant changes in sperm chromatin nuclear decondensation (P<0.05). In same way differences on the swelling degree was found assessed by measures in equatorial region of head sperm (P<0.05). Evaluation with rodamine-labeled actin (0.2μM) showed two different patterns with differences in percentages before and after cryopreservation (P<0.001). F-actin stabilization constrained the equatorial segment of FS while this was not observed in TS. The data showed that the presence of early changes in sPT integrity and changes in the F-actin localization on TS may suggest the participation in F-actin in decondensation process and probably that the disruption of actin-PT interaction during freezing-thawing process could have far-reaching consequences for the subsequent fertility of spermatozoa.

  20. The classic EDCs, phthalate esters and organochlorines, in relation to abnormal sperm quality: a systematic review with meta-analysis

    PubMed Central

    Wang, Chao; Yang, Lu; Wang, Shu; Zhang, Zhan; Yu, Yongquan; Wang, Meilin; Cromie, Meghan; Gao, Weimin; Wang, Shou-Lin

    2016-01-01

    The association between endocrine disrupting chemicals (EDCs) and human sperm quality is controversial due to the inconsistent literature findings, therefore, a systematic review with meta-analysis was performed. Through the literature search and selection based on inclusion criteria, a total of 9 studies (7 cross-sectional, 1 case-control, and 1 pilot study) were analyzed for classic EDCs (5 studies for phthalate esters and 4 studies for organochlorines). Funnel plots revealed a symmetrical distribution with no evidence of publication bias (Begg’s test: intercept = 0.40; p = 0.692). The summary odds ratios (OR) of human sperm quality associated with the classic EDCs was 1.67 (95% CI: 1.31–2.02). After stratification by specific chemical class, consistent increases in the risk of abnormal sperm quality were found in phthalate ester group (OR = 1.52; 95% CI: 1.09–1.95) and organochlorine group (OR = 1.98; 95% CI: 1.34–2.62). Additionally, identification of official data, and a comprehensive review of the mechanisms were performed, and better elucidated the increased risk of these classic EDCs on abnormal sperm quality. The present systematic review and meta-analysis helps to identify the impact of classic EDCs on human sperm quality. However, it still highlights the need for additional epidemiological studies in a larger variety of geographic locations. PMID:26804707

  1. The classic EDCs, phthalate esters and organochlorines, in relation to abnormal sperm quality: a systematic review with meta-analysis

    NASA Astrophysics Data System (ADS)

    Wang, Chao; Yang, Lu; Wang, Shu; Zhang, Zhan; Yu, Yongquan; Wang, Meilin; Cromie, Meghan; Gao, Weimin; Wang, Shou-Lin

    2016-01-01

    The association between endocrine disrupting chemicals (EDCs) and human sperm quality is controversial due to the inconsistent literature findings, therefore, a systematic review with meta-analysis was performed. Through the literature search and selection based on inclusion criteria, a total of 9 studies (7 cross-sectional, 1 case-control, and 1 pilot study) were analyzed for classic EDCs (5 studies for phthalate esters and 4 studies for organochlorines). Funnel plots revealed a symmetrical distribution with no evidence of publication bias (Begg’s test: intercept = 0.40 p = 0.692). The summary odds ratios (OR) of human sperm quality associated with the classic EDCs was 1.67 (95% CI: 1.31–2.02). After stratification by specific chemical class, consistent increases in the risk of abnormal sperm quality were found in phthalate ester group (OR = 1.52 95% CI: 1.09–1.95) and organochlorine group (OR = 1.98 95% CI: 1.34–2.62). Additionally, identification of official data, and a comprehensive review of the mechanisms were performed, and better elucidated the increased risk of these classic EDCs on abnormal sperm quality. The present systematic review and meta-analysis helps to identify the impact of classic EDCs on human sperm quality. However, it still highlights the need for additional epidemiological studies in a larger variety of geographic locations.

  2. The classic EDCs, phthalate esters and organochlorines, in relation to abnormal sperm quality: a systematic review with meta-analysis.

    PubMed

    Wang, Chao; Yang, Lu; Wang, Shu; Zhang, Zhan; Yu, Yongquan; Wang, Meilin; Cromie, Meghan; Gao, Weimin; Wang, Shou-Lin

    2016-01-25

    The association between endocrine disrupting chemicals (EDCs) and human sperm quality is controversial due to the inconsistent literature findings, therefore, a systematic review with meta-analysis was performed. Through the literature search and selection based on inclusion criteria, a total of 9 studies (7 cross-sectional, 1 case-control, and 1 pilot study) were analyzed for classic EDCs (5 studies for phthalate esters and 4 studies for organochlorines). Funnel plots revealed a symmetrical distribution with no evidence of publication bias (Begg's test: intercept = 0.40; p = 0.692). The summary odds ratios (OR) of human sperm quality associated with the classic EDCs was 1.67 (95% CI: 1.31-2.02). After stratification by specific chemical class, consistent increases in the risk of abnormal sperm quality were found in phthalate ester group (OR = 1.52; 95% CI: 1.09-1.95) and organochlorine group (OR = 1.98; 95% CI: 1.34-2.62). Additionally, identification of official data, and a comprehensive review of the mechanisms were performed, and better elucidated the increased risk of these classic EDCs on abnormal sperm quality. The present systematic review and meta-analysis helps to identify the impact of classic EDCs on human sperm quality. However, it still highlights the need for additional epidemiological studies in a larger variety of geographic locations.

  3. Sperm exposure to carbon-based nanomaterials causes abnormalities in early development of purple sea urchin (Paracentrotus lividus).

    PubMed

    Mesarič, Tina; Sepčić, Kristina; Drobne, Damjana; Makovec, Darko; Faimali, Marco; Morgana, Silvia; Falugi, Carla; Gambardella, Chiara

    2015-06-01

    We examined egg fertilisation in purple sea urchin (Paracentrotus lividus) after sperm exposure to carbon-based nanomaterials, carbon black (CB) and graphene oxide (GO), from 0.0001 mg/L to 1.0mg/L. Gastrula stage embryos were investigated for acetylcholinesterase and propionylcholinesterase activities, and their morphological characteristics. Plutei were analysed for morphological abnormalities, with emphasis on skeletal rod formation. Egg fertilisation was significantly affected by CB, at all concentrations tested. Loss of cell adhesion at the gastrula surface was observed in eggs fertilised with sperm treated with CB. However, concentration-dependent morphological anomalies were observed in the gastrulae and plutei formed after sperm exposure to either CB or GO. The activities of both cholinesterases decreased in the gastrulae, although not in a concentration-dependent manner. These effects appear to arise from physical interactions between these carbon-based nanomaterials and the sperm, whereby nanomaterials attached to the sperm surface interfere with fertilisation, which leads to disturbances in the signalling pathways of early embryonic development. Reduced cholinesterase activity in gastrulae from eggs fertilised with nanomaterial-treated sperm confirms involvement of the cholinergic system in early sea urchin development, including skeletogenesis.

  4. Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia.

    PubMed

    Piasecka, M; Gaczarzewicz, D; Laszczyńska, M; Starczewski, A; Brodowska, A

    2007-01-01

    Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

  5. An evidence-based approach to medicinal plants for the treatment of sperm abnormalities in traditional Persian medicine.

    PubMed

    Tahvilzadeh, M; Hajimahmoodi, M; Toliyat, T; Karimi, M; Rahimi, R

    2016-10-01

    Infertility is defined as inability of a sexually active couple to conceive after 1 year of regular intercourse without contraception. Male factors account for 20%-50% of cases of infertility. The aim of this study was to review medicinal plants that proposed to improve sperm abnormalities in traditional Persian medicine. For this purpose, PubMed, Scopus, GoogleScholar and Cochrane library were explored for medicinal plants used in traditional Persian medicine for sperm abnormalities to obtain studies giving any evidence for their efficacy and pharmacological mechanisms related to male infertility. Data were collected for the years 1966 to March 2015. For some of them, including Chlorophytum borivilianum, Crocus sativus, Nigella sativa, Sesamum indicum, Tribulus terrestris, Mucuna pruriens and Withania somnifera, more reliable evidence was found. The mechanisms involved in the beneficial effects of medicinal plants in sperm abnormalities are antioxidant, anti-inflammatory, anti-oedematous and venotonic activity as well as containing precursors for sperm production and increasing blood testosterone level. Various phytochemical categories including saponins, phytosterols, carotenoids, oxygenated volatile compounds, phenolic compounds and alkaloids seem to be responsible for these beneficial effects. Further studies are recommended for obtaining more conclusive results about the efficacy and safety of the mentioned medicinal plants.

  6. Increases in morphologically abnormal sperm in rats exposed to Co60 irradiation.

    PubMed

    Lock, L F; Soares, E R

    1980-01-01

    We have investigated the effects of testicular exposure to different doses of Co60 radiation on sperm morphology in F-344 rats. The results indicate that from 150 rad to 500 rad gamma irradiation causes statistically significant, dose-related increased in 1) the percent of morphologically aberrant sperm and 2) the frequency of tailless sperm. Both of these effects were detectable in sperm which were derived from treated spermatid, spermatocytes, and spermatogonial cells. These data indicate that the development of a sperm morphology assay in rats is feasible.

  7. The structure of sea-urchin-sperm histone phi 1 (H1) in chromatin and in free solution. Trypsin digestion and spectroscopic studies.

    PubMed

    Puigdomenech, P; Palau, J; Crane-Robinson, C

    1980-02-01

    The lysine-rich H1-type histone phi 1 from the sperm of the sea urchin Arbacia lixula has been subjected to tryptic digestion in free solution at high ionic strength. Tw trypsin-resistant peptides G phi 1 and LG phi 1 have been isolated, comprising 81 and 95 amino acids respectively, i.e. about a third of the intact histone. Circular dichroism and 270-MHz proton NMR have been used to demonstrate that the smaller peptide G phi 1 contains all the secondary and tertiary structure of intact histone phi 1. It is concluded that the resistant peptides represent a compact folded portion of the histone phi 1 chain, whilst the remaining two-thirds is disordered in free solution. Trypsin digestion of Arbacia lixula nuclei, under conditions where histone phi 1 remains tightly bound to the chromatin, leads to the same resistant peptide G phi 1. From this it is concluded that in chromatin the chain region corresponding to G phi 1 is likewise folded and inaccessible to trypsin, whilst the remaining exposed residues are located periferally and probably in an extended conformation. The rather unusual H1-type histone phi 1 from sea urchin sperm thus has a three-domain structure like calf thymus H1 and chicken erythrocyte H5. The present data emphasise the fact that this three-domain structure exists in chromatin and is not merely a free-solution artefact.

  8. Abnormal Sperm Development in pcd3J-/- Mice: the Importance of Agtpbp1 in Spermatogenesis

    PubMed Central

    Kim, Nameun; Xiao, Rui; Choi, Hojun; Kim, Jin-Hoi; Sang-Jun, Uhm; Chankyu, Park

    2011-01-01

    Homozygous Purkinje cell degeneration (pcd) mutant males exhibit abnormal sperm development. Microscopic examination of the testes from pcd3J-/- mice at postnatal days 12, 15, 18 and 60 revealed histological differences, in comparison to wild-type mice, which were evident by day 18. Greatly reduced numbers of spermatocytes and spermatids were found in the adult testes, and apoptotic cells were identified among the differentiating germ cells after day 15. Our immunohistological analysis using an antihuman AGTPBP1 antibody showed that AGTPBP1 was expressed in spermatogenic cells between late stage primary spermatocytes and round spermatids. A global gene expression analysis from the testes of pcd3J-/- mice showed that expression of cyclin B3 and de-ubiquitinating enzymes USP2 and USP9y was altered by >1.5-fold compared to the expression levels in the wild-type. Our results suggest that the pcd mutant mice have defects in spermatogenesis that begin with the pachytene spermatocyte stage and continue through subsequent stages. Thus, Agtpbp1, the gene responsible for the pcd phenotype, plays an important role in spermatogenesis and is important for survival of germ cells at spermatocytes stage onward. PMID:21110128

  9. Pronuclear epigenetic modification of protamine deficient human sperm following injection into mouse oocytes.

    PubMed

    Rajabi, Hoda; Mohseni-Kouchesfehani, Homa; Mohammadi-Sangcheshmeh, Abdollah; Farifteh-Nobijari, Fattaneh; Salehi, Mohammad

    2016-01-01

    Epigenetic abnormalities and abnormal chromatin structure in sperm may lead to male infertility. Protamine deficiency is among the disorders of chromatin structure in sperm. The study of epigenetic changes in male pronuclei is necessary since abnormal sperm is sometimes used to create embryos using assisted reproductive techniques. The present study was carried out to compare epigenetic global marks in male pronuclei derived from normal and protamine deficient sperm cells. To do so, interspecies fertilization was used to obtain the male pronucleus. Normal and protamine deficient sperm cells, which were identified by chromomycin A3 staining, were injected into mouse oocytes. Oocytes were cultured until pronuclear formation and were then labeled with different antibodies (anti 5-methylcytosine, anti 5-hydroxymethylcytosine, and anti acetyl H4K12). Based on the fluorescence intensity, the level of each of these epigenetic factors was determined and they revealed a significant relationship between the level of sperm protamine deficiency and sperm epigenetic factors. Protamine deficiency was found to be associated with an increased methylation (p=0) and decreased hydroxymethylation rate (p=0.015) of the male pronucleus chromatin. However, no association was found between protamine deficiency and the level of H4K12 acetylation (p=0.548). Also, the efficiency of fertilization in protamine deficient sperm cells was less than normal. These results suggest that protamine deficient sperm cells lead to the formation of epigenetically altered pronuclei.

  10. Age-related sperm DNA methylation changes are transmitted to offspring and associated with abnormal behavior and dysregulated gene expression.

    PubMed

    Milekic, M H; Xin, Y; O'Donnell, A; Kumar, K K; Bradley-Moore, M; Malaspina, D; Moore, H; Brunner, D; Ge, Y; Edwards, J; Paul, S; Haghighi, F G; Gingrich, J A

    2015-08-01

    Advanced paternal age (APA) has been shown to be a significant risk factor in the offspring for neurodevelopmental psychiatric disorders, such as schizophrenia and autism spectrum disorders. During aging, de novo mutations accumulate in the male germline and are frequently transmitted to the offspring with deleterious effects. In addition, DNA methylation during spermatogenesis is an active process, which is susceptible to errors that can be propagated to subsequent generations. Here we test the hypothesis that the integrity of germline DNA methylation is compromised during the aging process. A genome-wide DNA methylation screen comparing sperm from young and old mice revealed a significant loss of methylation in the older mice in regions associated with transcriptional regulation. The offspring of older fathers had reduced exploratory and startle behaviors and exhibited similar brain DNA methylation abnormalities as observed in the paternal sperm. Offspring from old fathers also had transcriptional dysregulation of developmental genes implicated in autism and schizophrenia. Our findings demonstrate that DNA methylation abnormalities arising in the sperm of old fathers are a plausible mechanism to explain some of the risks that APA poses to resulting offspring.

  11. Relationship between abnormal sperm morphology induced by dietary zinc deficiency and lipid composition in testes of growing rats.

    PubMed

    Merrells, Krystal J; Blewett, Heather; Jamieson, Jennifer A; Taylor, Carla G; Suh, Miyoung

    2009-07-01

    The present study investigated the effect of dietary Zn deficiency during sexual maturation on sperm integrity and testis phospholipid fatty acid composition. Male weanling Sprague-Dawley rats were randomised into four dietary groups for 3 weeks: Zn control (ZC; 30 mg Zn/kg); Zn marginally deficient (ZMD; 9 mg Zn/kg); Zn deficient (ZD; < 1 mg Zn/kg); pair fed (PF; 30 mg Zn/kg) to the ZD group. Morphology of cauda epididymal sperm and lipid profiles of testis phospholipids were analysed. The rats fed the ZD diet had a lower testis weight (P < 0.02). Seminal vesicles and prostate weight were also lower in the ZD and PF groups. Rats fed the ZD diet, but not the ZMD diet, had 34-35 % more abnormal spermatozoa and 24 % shorter sperm tail length than the ZC and PF rats (P < 0.001). Testis cholesterol concentration was higher in the ZD rats compared with the ZC and PF rats (P < 0.04). Testes were highly enriched with n-6 fatty acids by showing n-6 : n-3 fatty acid ratios of 27:1 in phosphatidylcholine (PC) and 23:1 in phosphatidylethanolamine (PE). The dominant fatty acid in testes was docosapentaenoic acid (22 : 5n-6), comprising 15 and 24 % of PC and PE, respectively. This fatty acid was significantly lower in the ZD rats, whereas 18 : 2n-6 was higher compared with the rats in the other diet groups. These results demonstrate that severe Zn deficiency adversely affects sperm integrity and modulates testis fatty acid composition by interrupting essential fatty acid metabolism. This suggests that Zn deficiency-associated abnormal testicular function is perhaps preceded by altered membrane fatty acid composition, especially of a major fatty acid, 22 : 5n-6.

  12. Cryopreservation of human spermatozoa decreases the number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation.

    PubMed

    Boitrelle, Florence; Albert, Martine; Theillac, Claire; Ferfouri, Fatma; Bergere, Marianne; Vialard, François; Wainer, Robert; Bailly, Marc; Selva, Jacqueline

    2012-01-01

    Even though cryopreservation of human spermatozoa is known to alter sperm motility and viability, it may also induce nuclear damages. The present study set out to determine whether or not cryopreservation alters motile sperm morphology under high magnification and/or is associated with chromatin decondensation. For 25 infertile men, we used high-magnification microscopy to determine the proportions of various types of motile spermatozoa before and after freezing-thawing: morphometrically normal spermatozoa with no vacuole (grade I), ≤ 2 small vacuoles (grade II), at least 1 large vacuole or >2 small vacuoles (grade III), and morphometrically abnormal spermatozoa (grade IV). The spermatozoa's chromatin condensation and viability were also assessed before and after freezing-thawing. Cryopreservation induced sperm nuclear vacuolization. It decreased the proportion of grade I + II spermatozoa (P < .001). It induced a decrease in the sperm viability rate (P < .001) and increased the proportion of sperm with noncondensed chromatin (P < .001). The latter parameter was strongly correlated with sperm viability (r = 0.71; P < .001). However, even motile sperm presented a failure of chromatin condensation after freezing-thawing, because the proportion of sperm with noncondensed chromatin was correlated with high-magnification morphology (r = -0.49 and 0.49 for the proportions of grade I + II and grades III + IV, respectively; P < .001). Cryopreservation alters the organelle morphology of motile human spermatozoa and induces sperm chromatin decondensation. High-magnification microscopy may be useful for evaluating frozen-thawed spermatozoa before use in assisted reproductive technology procedures (such as intrauterine insemination, in vitro fertilization, and intracytoplasmic sperm injection) and for performing research on cryopreservation methods. If frozen-thawed sperm is to be used for intracytoplasmic sperm injection, morphological selection under high magnification may

  13. Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation.

    PubMed

    Chohan, K R; Griffin, J T; Carrell, D T

    2004-10-01

    Staining of cells with acridine orange (AO) has been widely accepted as a predictor of DNA damage in many cell types. Because of variability of protocols used in previous studies, the AO staining technique has not been widely accepted as a screening test to predict DNA damage in human sperm. In order to further validate the use of AO staining, sperm were evaluated using numerous variations in the staining protocol. This study also elucidated the effects of cryopreservation on sperm DNA. Sperm fixation in Carnoy's solution showed significantly (P < 0.05) more DNA damage (29.9 +/- 4.5%) than 2% glutaraldehyde (14.4 +/- 2.1%), 4% paraformaldehyde (5.5 +/- 1.7%), no fixation (15.8 +/- 4.3%) but did not differ from Diff Quik solution (19.2 +/- 5.8%). No difference was observed for sperm DNA damage assessment using a 0.2 m (15.5 +/- 3.2%) or 0.3 m (14.9 +/- 3.3%) concentration of Na(2)HPO(4).7H(2)O in the AO staining solution. Frozen-thawed semen samples showed increased damage to sperm DNA under both Carnoy's (fresh: 10.9 +/- 1.3%; frozen: 30.8 +/- 2.9%; P < 0.05) and Diff Quik fixation (fresh: 6.2 +/- 0.8; frozen: 17.1 +/- 2.5%P < 0.05). Present data also showed that spermatozoa from some individuals are more prone to DNA damage after freezing and thawing procedures than others. In conclusion, Carnoy's fixative provides a better predictive value for DNA damage to sperm using AO staining. Additionally, cryopreservation increased damage to the sperm DNA.

  14. Remodeling sperm chromatin in Xenopus laevis egg extracts: the role of core histone phosphorylation and linker histone B4 in chromatin assembly

    PubMed Central

    1994-01-01

    We find that the remodeling of the condensed Xenopus laevis sperm nucleus into the paternal pronucleus in egg extracts is associated with phosphorylation of the core histones H2A, H2A.X and H4, and uptake of a linker histone B4 and a HMG 2 protein. Histone B4 is required for the assembly of chromatosome structures in the pronucleus. However neither B4 nor core histone phosphorylation are required for the assembly of spaced nucleosomal arrays. We suggest that the spacing of nucleosomal arrays is determined by interaction between adjacent histone octamers under physiological assembly conditions. PMID:8045925

  15. Comparison of semen variables, sperm DNA damage and sperm membrane proteins in two male layer breeder lines.

    PubMed

    M, Shanmugam; T R, Kannaki; A, Vinoth

    2016-09-01

    Semen variables are affected by the breed and strain of chicken. The present study was undertaken to compare the semen quality in two lines of adult chickens with particular reference to sperm chromatin condensation, sperm DNA damage and sperm membrane proteins. Semen from a PD3 and White Leghorn control line was collected at 46 and 47 weeks and 55 weeks of age. The semen was evaluated for gross variables and sperm chromatin condensation by aniline blue staining. Sperm DNA damage was assessed by using the comet assay at 47 weeks of age and sperm membrane proteins were assessed at 55 weeks of age. The duration of fertility was studied by inseminating 100 million sperm once into the hens of the same line as well as another line. The eggs were collected after insemination for 15days and incubated. The eggs were candled on 18th day of incubation for observing embryonic development. The White Leghorn control line had a greater sperm concentration and lesser percentage of morphologically abnormal sperm at the different ages where assessments occurred. There was no difference in sperm chromatin condensation, DNA damage and membrane proteins between the lines. Only low molecular weight protein bands of less than 95kDa were observed in samples of both lines. The line from which semen was used had no effect on the duration over which fertility was sustained after insemination either when used in the same line or another line. Thus, from the results of the present study it may be concluded that there was a difference in gross semen variables between the lines that were studied, however, the sperm chromatin condensation, DNA damage, membrane proteins and duration over which fertility was sustained after insemination did not differ between the lines.

  16. Sperm abnormalities induced by pre-pubertal exposure to cyclophosphamide are effectively mitigated by Moringa oleifera leaf extract.

    PubMed

    Nayak, G; Vadinkar, A; Nair, S; Kalthur, S G; D'Souza, A S; Shetty, P K; Mutalik, S; Shetty, M M; Kalthur, G; Adiga, S K

    2016-03-01

    Moringa oleifera L. is a medicinal plant with potential antioxidant property. This study was aimed at investigating the chemoprotective effect of Moringa oleifera leaf extract (MOE) on cyclophosphamide (CP)-induced testicular toxicity. Two-week-old male Swiss albino mice were intraperitoneally injected with phosphate-buffered saline, 50 mg kg(-1) of CP and 25 mg kg(-1) of MOE. In combination treatment, mice were injected with 25 mg kg(-1) of MOE 24 h prior to CP injection, 24 h prior and post-CP injection and 24 h post-CP injection for 5 consecutive days (10 mg kg(-1) ). Six weeks later, mice were sacrificed to assess epididymal sperm parameters. MOE alone did not have any significant effect on sperm parameters. However, acute injection of CP resulted in significant decline in motility (P < 0.001), increase in head abnormality (P < 0.01) and DNA damage (P < 0.05). Combining MOE with CP increased the sperm density, motility and reduced head defect and DNA damage, irrespective of the schedule and dosage of MOE. Administration of MOE prior to CP significantly elevated the level of superoxide dismutase and catalase with concomitant decrease in lipid peroxidation in the testicular tissue. In conclusion, MOE may have potential benefit in reducing the loss of male gonadal function following chemotherapy.

  17. Evidence that single-stranded DNA breaks are a normal feature of koala sperm chromatin, while double-stranded DNA breaks are indicative of DNA damage.

    PubMed

    Zee, Yeng Peng; López-Fernández, Carmen; Arroyo, F; Johnston, Stephen D; Holt, William V; Gosalvez, Jaime

    2009-08-01

    In this study, we have used single and double comet assays to differentiate between single- and double-stranded DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa. We have also investigated the likelihood that single-stranded DNA breakage is part of the natural spermiogenic process in koalas, where its function would be the generation of structural bends in the DNA molecule so that appropriate packaging and compaction can occur. Koala spermatozoa were examined using the sperm chromatin dispersion test (SCDt) and comet assays to investigate non-orthodox double-stranded DNA. Comet assays were conducted under 1) neutral conditions; and 2) neutral followed by alkaline conditions (double comet assay); the latter technique enabled simultaneous visualisation of both single-stranded and double-stranded DNA breaks. Following the SCDt, there was a continuum of nuclear morphotypes, ranging from no apparent DNA fragmentation to those with highly dispersed and degraded chromatin. Dispersion morphotypes were mirrored by a similar diversity of comet morphologies that could be further differentiated using the double comet assay. The majority of koala spermatozoa had nuclei with DNA abasic-like residues that produced single-tailed comets following the double comet assay. The ubiquity of these residues suggests that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with 'true' DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA with a diffuse single tail to nuclei that exhibited both single- and double-stranded breaks with two comet tails.

  18. The sequential appearance of sperm abnormalities after scrotal insulation or dexamethasone treatment in bulls.

    PubMed Central

    Barth, A D; Bowman, P A

    1994-01-01

    Scrotal insulation and dexamethasone treatment were used as a model to compare the effect of testicular heating and stress on spermatogenesis. Insulation was applied to the scrotum of eight bulls (insulated) for a period of four days, eight bulls were treated daily for seven days with 20 mg dexamethasone injected intramuscularly, and four bulls were untreated controls. Semen from four bulls in each group was collected and evaluated over a six-week period after treatment. Blood samples for testosterone analysis were taken hourly for eight hours at the beginning and the end of the six-week period from the control bulls and before and after treatment from the four insulated and four dexamethasone-treated bulls that were not used for semen collection. At the end of the last blood sampling period, the four bulls in each group were castrated for the collection of testicular tissue for the determination of testosterone concentrations. Basal, peak episodic, and mean serum testosterone concentrations among control bulls, pre and postinsulated bulls, and pretreatment samples of dexamethasone-treated bulls were not different (p > 0.05); however, bulls that had received dexamethasone treatments had significantly lower basal, peak episodic, and mean testosterone concentrations (p < 0.05). Tissue concentrations of testosterone in control, insulated, and dexamethasone-treated bulls were not significantly different but tended to be lower in dexamethasone-treated bulls (p > 0.13). The spermiograms of the control bulls varied insignificantly over the six-week sampling period; however, there was a marked increase in sperm defects in insulated and dexamethasone-treated bulls. The types of sperm defects and the temporal relationships of rises and declines of sperm defects were quite similar for both treatments. All bulls recovered to approximately pretreatment levels of sperm defects by six weeks after the initiation of treatment. Results indicate that two of the most common types of

  19. Structure of chromatin in spermatozoa.

    PubMed

    Björndahl, Lars; Kvist, Ulrik

    2014-01-01

    The specialized structure of the sperm chromatin has a dual function - first to protect the DNA from damage during storage and transport to the oocyte, and then to enable a rapid and complete unpacking of the undamaged paternal genome in the ooplasm. It is evident that zinc has a pivotal role in maintaining the structural stability and in enabling a rapid decondensation at the appropriate time. It is important for the sperm chromatin structure that the spermatozoa are ejaculated together with the zinc-rich prostatic secretion. Early exposure to zinc-binding seminal vesicular fluid can deplete the sperm chromatin of zinc and most likely induce surplus formation of disulfide bridges, likely to cause incomplete and delayed decondensation of the sperm chromatin in the oocyte. A premature decrease in sperm chromatin structure stability is likely to increase the risk for damage to the DNA due to increased access to the genome for DNA damaging compounds. The status of the sperm chromatin structure can vary in vitro depending on the exposure to zinc-depleting conditions when spermatozoa are stored in semen after ejaculation. When sperm DNA damage tests are evaluated and validated, it is therefore essential to also take into account the dynamics of zinc-dependent and zinc-independent sperm chromatin stability.

  20. Disruption of dmc1 Produces Abnormal Sperm in Medaka (Oryzias latipes)

    PubMed Central

    Chen, Ji; Cui, Xiaojuan; Jia, Shaoting; Luo, Daji; Cao, Mengxi; Zhang, Yunsheng; Hu, Hongling; Huang, Kaiyao; Zhu, Zuoyan; Hu, Wei

    2016-01-01

    DMC1 is a recombinase that is essential for meiotic synapsis. Experiments in extensive species of eukaryotes have indicated the independent role of DMC1 in repairing double strand breaks (DSBs) produced during meiosis I. Mutation of dmc1 in mice and human often leads to obstacles in spermatogenesis and male sterility. Here, we report on the disruption of dmc1 in male medaka (Oryzias latipes). Synapsis was disturbed in the mutant medaka testis nuclei, as observed in mice and other organisms. Unexpectedly, the mutant medaka could produce a few sperm and, although most of these had multiple tail or multiple head malformations, some of them could swim, and few of them even had insemination ability. Our transcriptome analysis showed that there was not a remarkable change in the expression of most of the genes involved in the pathways associated with the meiotic DNA repair and flagella assembly. Our results provided an indication of the accessory mechanisms that might be involved in the repair of DSBs during meiosis. In a species besides humans, we provided evidence that disorders in meiosis recombination might lead to the malformation of sperm. PMID:27480068

  1. Mouse organ coefficient and abnormal sperm rate analysis with exposure to tap water and source water in Nanjing reach of Yangtze River.

    PubMed

    Zhang, Rongfei; Zhang, Liujun; Jiang, Dongsheng; Zheng, Kai; Cui, Yibin; Li, Mei; Wu, Bing; Cheng, Shupei

    2014-05-01

    Organ coefficients (including kidney, testis, liver and spleen coefficient) and abnormal sperm rate were used in our study to reflect the exposure to the Yangzte River water. The concentrations of total dissolved metals and semi-volatile organic compounds in tap and source water were measured by ICP-OES and GC-MS, respectively. After mice were fed with purified water (CK), Nanjing tap water (NJT) and Nanjing source water (NJS) for 90 day, the individual and organs (including kidney, testis, liver and spleen) of each mouse were weighted. And abnormal sperm types (such as hook less, banana-like form, amorphous, folded and two tails) were determined by microscope. The results showed that significant differences of liver coefficient between experimental group (NJT, NJS) and control group (CK) were observed; furthermore liver coefficient is positive correlation with the concentrations of total dissolved metals. However, no significant differences of abnormal sperm rates between experimental group (NJT, NJS) and control group (CK) were noted. So liver coefficient might be more sensitive than other organ coefficients to reflect the exposure to tap water and source water, while abnormal sperm rate could not be used to reveal the exposure to them.

  2. Safety evaluation of a triazine compound nitromezuril by assessing bacterial reverse mutation, sperm abnormalities, micronucleus and chromosomal aberration.

    PubMed

    Fei, Chenzhong; Zhang, Jie; Lin, Yang; Wang, Xiaoyang; Zhang, Keyu; Zhang, Lifang; Zheng, Wenli; Wang, Mi; Li, Tao; Xiao, Sui; Xue, Feiqun; Wang, Chunmei

    2015-04-01

    Nitromezuril (NZL) is a novel triazine compound that exhibits remarkable anticoccidial activity. However, mutagenicity and genotoxicity of NZL have not been evaluated to date. This study evaluated the potential risks of NZL by testing for bacterial reverse mutation (Ames), mouse sperm abnormality (SA), bone marrow micronucleus (MN) and chromosomal aberration (CA). Mice were orally administered with NZL at 385, 192 and 96 mg/kg, corresponding to 0.5 ×, 0.25 × and 0.125 × the LD50 of NZL, respectively. No significant increases in SA and CA were found in mice treated with NZL for 5d and 3d, respectively (P>0.05). NZL at 96-385 mg/kg did not have significant influence on micronucleated polychromatic erythrocyte counts (P>0.05). These results suggest that NZL is not genotoxic. However, Ames test results were positive both with and without the S9 system for Salmonella typhimurium TA98 and TA100, suggesting that NZL may be mutagenic. The mutagenic effects of NZL were different in in vitro and in vivo assays. Further studies should be conducted to confirm the safety of using and developing NZL as a novel anticoccidial drug.

  3. Paternal monoenergetic neutron exposure results in abnormal sperm, and embryonal lethality and transgenerational tumorigenesis in mouse F1 offspring.

    PubMed

    Watanabe, Hiromitsu; Toyoshima, Megumi; Ishikawa, Masayori; Kamiya, Kenji

    2010-05-01

    Experiments were conducted to assay whether monoenergetic neutron-induced genetic damage in parental germline cells can give rise to development of cancer in the offspring. Seven-week-old C3H male mice were irradiated with monoenergetic neutrons with energy levels of 0.2 or 0.6 MeV at doses of 0, 50, 100 or 200 cGy. Two weeks after irradiation, when the male mice showed an increased incidence of sperm abnormalities, they were mated with virgin 9-week-old C57BL females. Litter size was decreased and embryo lethalities were increased in a dose-dependent manner. Furthermore, tumor incidence in male offspring born to male mice irradiated with 25 or 50 cGy at 0.6 MeV showed a tendency for increase as compared to the non-irradiated group value. Liver tumors in the 50 cGy group were significantly increased (P=0.03). It is concluded that the increased hepatic tumor risk in the F1 generation may have been caused by genetic transmission of some hepatoma-associated trait(s) induced by monoenergetic neutron irradiation.

  4. Vacuoles in sperm head are not associated with head morphology, DNA damage and reproductive success.

    PubMed

    Fortunato, Adriana; Boni, Raffaele; Leo, Rita; Nacchia, Giuseppina; Liguori, Francesca; Casale, Sofia; Bonassisa, Paolo; Tosti, Elisabetta

    2016-02-01

    In this retrospective study of 873 men enrolled for assisted reproduction techniques, relationships between sperm quality parameters, motile sperm organelle morphology examination (MSOME), DNA damage and live birth rate were evaluated. The presence of vacuoles in the sperm heads was detected by MSOME. Either chromatin decondensation or DNA fragmentation was used to study DNA damage. Results show that age significantly affected some of the examined parameters. In particular, sperm concentration was positively correlated (R = 0.088; P = 0.01) and chromatin decondensation was negatively correlated (R = -0.102; P = 0.003) with age. Furthermore, live birth rate was significantly lower in men aged 40 years or older (P < 0.02) compared with the younger age groups. The presence of sperm head vacuoles was not associated with head morphology, main sperm quality parameters, DNA fragmentation and live birth rate. Considering sperm heads in relation to the shape (normal/abnormal) and vacuoles (presence/absence), no significant variations in the occurrence of vacuoles in either normal or abnormal heads were found. These data suggest that vacuoles are physiological features that do not alter sperm functionality, and it seems that MSOME is not necessary for increasing the success of assisted reproduction techniques.

  5. Relationship between DNA damage in sperm after ex vivo exposure and abnormal embryo development in the progeny of the three-spined stickleback.

    PubMed

    Santos, R; Palos-Ladeiro, M; Besnard, A; Porcher, J M; Bony, S; Sanchez, W; Devaux, A

    2013-04-01

    Many xenobiotics released in the aquatic environment exhibit a genotoxic potential toward organisms. Long term exposure to such compounds is expected to lead to multigenerational reproductive defects, further influencing the recruitment rate and hence, the population dynamics. Paternal exposure to genotoxicants was previously shown to increase abnormal development in the progeny of mammalian or aquatic species. The aim of this study was to evaluate the relationship between DNA damage in sperm of the fish three-spined stickleback and progeny developmental defects. Spermatozoa were exposed ex vivo to an alkylating agent (methyl methanesulfonate) before in vitro fertilization and DNA damage was assessed by the alkaline comet assay. A significant relationship between abnormal development and sperm DNA damage was underlined. This study illustrates the interest to use germ cell DNA damage after ex vivo exposure to evaluate the impact of genotoxic compounds on progeny fitness in aquatic organisms.

  6. Induction of micronuclei and sister chromatid exchange in bone-marrow cells and abnormalities in sperm of Algerian mice (Mus spretus) exposed to cadmium, lead and zinc.

    PubMed

    Tapisso, Joaquim Torres; Marques, Carla Cristina; Mathias, Maria da Luz; Ramalhinho, Maria da Graça

    2009-08-01

    As a consequence of human activities, large amounts of cadmium, lead and zinc are released in the environment, often simultaneously. The aim of this study was to investigate under experimental conditions the DNA damage induced in Algerian mice (Mus spretus) exposed to cadmium (Cd), lead (Pb) and zinc (Zn) separately, or in selected combinations. Three cytogenetic end points were considered: the frequencies of micronucleated cells (MN) and sister chromatid exchange (SCE) in the bone marrow and the frequency of sperm abnormalities. Mice were treated by intraperitoneal (i.p.) injections with 5 or 10 doses of aqueous solutions of cadmium acetate, lead acetate and zinc acetate in concentrations corresponding to 1/10 of the LD50, respectively, 21.5, 0.46 and 1.5 mg/kg bw. The control groups were injected in the same way with distilled water. With only one exception (Cd + Zn group treated with 5 doses), the results show a significant increase of MN in all groups for both treatments (5 and 10 doses). Similarly, the results concerning the SCE revealed a statistically significant increase in all treated animals, with the exception of the Zn group treated with 5 doses. The number of sperm abnormalities was significantly higher in animals treated with 5 doses, except in the group Pb + Zn. In animals treated with 10 doses the number of sperm abnormalities was always statistically higher compared with controls. This study indicates that cadmium, lead and zinc can induce MN, SCEs and sperm abnormalities in Algerian mice and that the clastogenic potential is dependent on the time of exposure and the interaction between the three elements, confirming the environmental damage that may result from the simultaneous action of several metals. Most relevant is the toxic potential for Zn, related with the dose, which may compromise its protective effect against other metal contaminations, such as cadmium.

  7. Detection of sex chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization

    SciTech Connect

    Wyrobek, A.J.; Robbins, W.A. |; Pinkel, D.; Weier, H.U.; Mehraein, Y. |

    1994-10-15

    Sex chromosome aneuploidy is the most common numerical chromosomal abnormality in humans at birth and a substantial portion of these abnormalities involve paternal chromosomes. An efficient method is presented for using air-dried smears of human semen to detect the number of X and Y chromosomes in sperm chromatin using two-chromosome fluorescence in situ hybridization. Air-dried semen smears were pre-treated with dithiothreitol and 3,4-diiodosalicylate salt to decondense the sperm chromatin and then were hybridized with repetitive sequence DNA probes that had been generated by PCR and differentially labeled. Hybridizations with X and Y specific probes showed the expected ratio of 50%X:50%Y bearing sperm. Sperm carrying extra fluorescence domains representing disomy for the X or Y chromosomes occurred at frequencies of {approximately} 4 per 10,000 sperm each. Cells carrying both X and Y fluorescence domains occurred at a frequency of {approximately} 6/10,000. Thus, the overall frequency of sperm that carried an extra sex chromosome was 1.4/1,000. The frequencies of sperm carrying sex chromosome aneuploidies determined by hybridization did not differ statistically from those reported from the same laboratory using the human-sperm/hamster-egg cytogenetic technique. Multi-chromosome fluorescence in situ hybridization to sperm is a promising method for assessing sex-ratio alterations in human semen and for determining the fraction of sperm carrying sex or other chromosome aneuploidies which may be transmissible to offspring. 44 refs., 1 fig., 3 tabs.

  8. Tales of the Tail and Sperm Head Aches Changing concepts on the prognostic significance of sperm pathologies affecting the head, neck and tail

    PubMed Central

    Chemes, Héctor E; Alvarez Sedo, Cristian

    2012-01-01

    This article presents an update on the variable prognostic significance of different sperm pathologies in patients with severe male factor infertility due to morphology and motility disorders. Severe asthenozoospermia is one of the leading causes of male infertility as spermatozoa cannot reach the oocyte and/or penetrate normally. Identifying structural causes of sperm immotility was of great concern before the advent of intracytoplasmic sperm injection (ICSI), because immotility was the limiting factor in the treatment of these patients. In these cases, in vitro methods are used to identify live spermatozoa or stimulate sperm motility to avoid selection of non-viable cells. With these advances, fertilization and pregnancy results have improved dramatically. The identification of genetic phenotypes in asthenozoospermia is important to adequately inform patients of treatment outcomes and risks. The one sperm characteristic that seriously affects fertility prognosis is teratozoospermia, primarily sperm head and neck anomalies. Defects of chromatin condensation and acrosomal hypoplasia are the two most common abnormalities in severe teratozoospermia. The introduction of microscopic methods to select spermatozoa and the development of new ones to evaluate sperm quality before ICSI will assure that ultrastructural identification of sperm pathologies will not only be of academic interest, but will also be an essential tool to inform treatment choice. Herein, we review the differential roles played by sperm components in normal fertilization and early embryo development and explore how assisted reproductive technologies have modified our concepts on the prognostic significance of sperm pathologies affecting the head, neck, mid-piece and tail. PMID:22198630

  9. Comparison between human sperm preservation medium and TEST-yolk buffer on protecting chromatin and morphology integrity of human spermatozoa in fertile and subfertile men after freeze-thawing procedure.

    PubMed

    Hammadeh, M E; Greiner, S; Rosenbaum, P; Schmidt, W

    2001-01-01

    The aim of this study was to identify the detrimental effect of the freeze-thaw process on chromatin integrity and morphology of human spermatozoa, and to determine whether human sperm preservation medium (HSPM) or TEST-yolk buffer (TYB) offers a better protection to spermatozoa from cryodamage after the freeze-thaw procedure. Thirty-five semen samples obtained from couples childless because of male factor infertility (subfertile men, group 1) and 25 semen samples from healthy, normal volunteers of proven fertility (group 2) were included in the study. Each semen sample was divided into 2 parts, the first part was mixed with HSPM and the other with TYB (1:1), and frozen with a controlled slow-stage freezer, before plunging into liquid nitrogen. Twelve smears from each semen sample were made before (n = 4) and after (n = 8) the freeze-thaw process. Chromatin structure was evaluated after staining using the acridine orange (AO) test, whereas morphology was analyzed according to strict criteria. The mean percentage of spermatozoa that exhibited normal morphology and intact chromatin structure was decreased after freeze-thaw in all samples treated with HSPM or TYB in comparison with the value observed in the native semen samples of both groups. However, TYB preserved chromatin and morphology significantly better than HSPM did (9.3% +/- 5.6% and 88.7% +/- 11.2% vs. 7.8% +/- 4.2% and 85.5% +/- 12.5%, respectively). Therefore, TYB could be recommended as a first choice cryoprotectant for semen preservation in order to avoid extra chromatin structure damage and morphology alterations of spermatozoa not only for patients pursuing assisted reproduction, but also for donor samples.

  10. Sperm protamine-status correlates to the fertility of breeding bulls.

    PubMed

    Dogan, Sule; Vargovic, Peter; Oliveira, Rodrigo; Belser, Lauren E; Kaya, Abdullah; Moura, Arlindo; Sutovsky, Peter; Parrish, John; Topper, Einko; Memili, Erdoğan

    2015-04-01

    During fertilization, spermatozoa make essential contributions to embryo development by providing oocyte activating factors, centrosomal components, and paternal chromosomes. Protamines are essential for proper packaging of sperm DNA; however, in contrast to the studies of oocyte-related female infertility, the influence of sperm chromatin structure on male infertility has not been evaluated extensively. The objective of this study was to determine the sperm chromatin content of bull spermatozoa by evaluating DNA fragmentation, chromatin maturity/protamination, PRM1 protein status, and nuclear shape in spermatozoa from bulls with different fertility. Relationships between protamine 1 (PRM1) and the chromatin integrity were ascertained in spermatozoa from Holstein bulls with varied (high vs. low) but acceptable fertility. Sperm DNA fragmentation and chromatin maturity (protamination) were tested using Halomax assay and toluidine blue staining, respectively. The PRM1 content was assayed using Western blotting and in-gel densitometry, flow cytometry, and immunocytochemistry. Fragmentation of DNA was increased and chromatin maturity significantly reduced in spermatozoa from low-fertility bulls compared to those from high-fertility bulls. Field fertility scores of the bulls were negatively correlated with the percentage of spermatozoa displaying reduced protamination and fragmented DNA using toluidine blue and Halomax, respectively. Bull fertility was also positively correlated with PRM1 content by Western blotting and flow cytometry. However, detection of PRM1 content by Western blotting alone was not predictive of bull fertility. In immunocytochemistry, abnormal spermatozoa showed either a lack of PRM1 or scattered localization in the apical/acrosomal region of the nuclei. The nuclear shape was distorted in spermatozoa from low-fertility bulls. In conclusion, we showed that inadequate amount and localization of PRM1 were associated with defects in sperm chromatin

  11. Abnormal sperm in mice with targeted deletion of the act (activator of cAMP-responsive element modulator in testis) gene

    PubMed Central

    Kotaja, Noora; De Cesare, Dario; Macho, Betina; Monaco, Lucia; Brancorsini, Stefano; Goossens, Ellen; Tournaye, Herman; Gansmuller, Anne; Sassone-Corsi, Paolo

    2004-01-01

    ACT [activator of cAMP-responsive element modulator (CREM) in testis] is a LIM-only protein that interacts with transcription factor CREM in postmeiotic male germ cells and enhances CREM-dependent transcription. CREM regulates many crucial genes required for spermatid maturation, and targeted mutation of the Crem gene in the mouse germ-line blocks spermatogenesis. Here we report the phenotype of mice in which targeted disruption of the act gene was obtained by homologous recombination. Whereas the seminiferous tubules of the act–/– mice contain all of the developmental stages of germ cells and the mice are fertile, the amount of mature sperm in the epididymis is drastically reduced. The residual sperm display severe abnormalities, including fully folded tails and aberrant head shapes. These results indicate that numerous postmeiotic genes under CREM control require the coactivator function of ACT. Thus, the fine-tuning of sperm development is achieved by the coordinated action of two transcriptional regulators. PMID:15247423

  12. Pathogenetic transition in the morphology of abnormal sperm in the testes and the caput, corpus, and cauda epididymides of male rats after treatment with 4,6-dinitro-o-cresol.

    PubMed

    Takahashi, Ken L; Takahashi, Naofumi; Hojo, Hitoshi; Kuwahara, Maki; Aoyama, Hiroaki; Teramoto, Shoji

    2006-10-01

    In order to elucidate the pathogenesis of tailless sperm, 4,6-dinitro-o-cresol (DNOC) was administered to Jcl:SD male rats at daily oral doses of 0, 10 or 15mg/kg for 5 days. Sperm were collected from the caput, corpus, and cauda epididymides on days 1, 7 and 14 after the last dosing (D1, D7 and D14, respectively), counted and examined morphologically by phase-contrast and scanning electron microscopy. The incidence of abnormal sperm was significantly increased in the DNOC 15mg/kg group. On D1, peeled sperm (loss of mitochondrial sheath at the proximal end of the middle piece) was frequently observed in the caput epididymides, whereas sperm in the corpus and cauda epididymides had normal morphology. Distribution of the peeled sperm changed as time passed and the corpus epididymides showed a peak incidence on D7. On D14, the highest incidence of abnormal sperm was observed in the cauda epididymides, where the major abnormality was tailless. Similar effects were also found in the 10mg/kg group but were less potent. Transmission electron microscopy of testicular sperm on D1 revealed the presence of elongated spermatids that lacked the mitochondrial sheath at the proximal end of the middle piece, although the round and elongating spermatids looked normal. These results suggest that DNOC exposure of male rats primarily causes partial loss of the mitochondrial sheath in the testicular elongated spermatids, and that the affected sperm become tailless by D14 after reaching the cauda epididymides.

  13. Efficient isolation of sperm with high DNA integrity and stable chromatin packaging by a combination of density-gradient centrifugation and magnetic-activated cell sorting

    PubMed Central

    Kwak, Su-Jin; Kim, Seok-Gi; Kim, Youn-Young; Park, Ji-Young; Yoo, Chang-Seok; Park, Il-Hae; Sun, Hong-Gil; Kim, Jae-Won; Lee, Kyeong-Ho

    2016-01-01

    Objective This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. Methods Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. Results The sperm DFI showed a significant correlation (r=–0.347, p<0.001) with sperm motility and morphology (r=–0.114, p<0.05) but not with other semen parameters. The DFI (11.5%±2.0%) of semen samples was significantly reduced by DGC (8.1%±4.1%) or MACS alone (7.4%±3.9%) (p<0.05). The DFI was significantly further reduced by a combination of DGC and MACS (4.1%±1.3%, p<0.05). Moreover, the combination of DGC and MACS (1.6%±1.1%, p<0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC (4.4%±3.2%) or MACS alone (3.4%±2.2%). Conclusion The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use. PMID:28090458

  14. Normal sperm morphology and changes of semen characteristics and abnormal morphological spermatozoa among peri-mating seasons in captive japanese black bears (Ursus thibetanus japonicus).

    PubMed

    Okano, Tsukasa; Murase, Tetsuma; Nakamura, Sachiko; Komatsu, Takeshi; Tsubota, Toshio; Asano, Makoto

    2009-04-01

    The objectives of this study were to obtain morphological data for normal spermatozoa and to investigate seasonal changes (the early, mid- and post-mating seasons) in abnormal morphology of spermatozoa and the characteristics of semen in Japanese black bears. Semen was collected by electroejaculation from 34 captive male Japanese black bears a total of 74 times. Length of head, width of head, length of midpiece and total length of the spermatozoa were 6.3 +/- 0.4, 4.5 +/- 0.3, 10.4 +/- 0.7 and 69.6 +/- 3.1 mum (mean +/- SD; 20 semen, 200 spermatozoa), respectively. In the semen collected during the mid-mating season, ejaculate volume, ejaculate pH, sperm concentration, total sperm count, motility, viability and intact acrosomes were 0.46 +/- 0.36 ml, 7.3 +/- 0.4, 659 +/- 644 x 10(6)/ml, 214 +/- 208 x 10(6), 82.9 +/- 9.6%, 89.3 +/- 9.5% and 97.0 +/- 3.2% (mean +/- SD; n=21, in ejaculate pH n=8), respectively. Sperm motility and viability in the early (n=7) and mid-mating (n=21) seasons were significantly higher than in the post-mating (n=8) season. The rates of detached heads in the early and mid-mating season were significantly lower than in the post-mating season. The main abnormal morphologies observed (mean +/- SD%; n=23) were simply bent tail (19.9 +/- 22.6), distal droplets (13.5 +/- 11.7), proximal droplets (9.6 +/- 7.8), teratoid spermatozoa (6.7 +/- 10.7), knobbed acrosome (4.9 +/- 8.6), acrosome damage (3.7 +/- 2.8) and bent midpiece (3.7 +/- 5.1). The data will be useful for artificial breeding and further research on male reproductive physiology in this species.

  15. Detection of alterations in human sperm using magnetic orientation techniques

    NASA Astrophysics Data System (ADS)

    Sakhnini, Lama; Dairi, Maheen; Manaa, Hacene

    2007-09-01

    In this study we report on magnetic orientation of human sperms. Samples were taken from 17 donors. Normal human sperms became oriented with their long axis perpendicular to the magnetic field ( 1 Tesla maximum). Total orientation was achieved with magnetic field at about one Tesla, while for abnormal sperms the magnetic behavior was different. The dependence of the measured degree of orientation on the intensity of the magnetic field was in good agreement with the theoretical equation for the magnetic orientation of diamagnetic substances. As a result for a numerical analysis based on the equation, the anisotropic diamagnetic susceptibility of normal sperm was found to be ▵ χ= 8×10 -20 J/T2. The degree of orientation was influenced by the alterations in the shape of the head, body or the tail. It has been suggested that the DNA in the sperm head retain the strong magnetic anisotropy to counter balance the magnetic anisotropy retained by flagellum microtubules. Recent studies demonstrated a well-defined nuclear architecture in human sperm nucleus, where the head morphology has significant correlation with sperm chromatin structure assay SCSA. Then as the methods to evaluate SCSA can be difficult and expensive our simple magnetic orientation technique can be an alternative to diagnose alteration in DNA.

  16. Sperm tail abnormalities in mutant mice with neo(r) gene insertion into an intron of the keratin 9 gene.

    PubMed

    Rivkin, Eugene; Eddy, Edward M; Willis, William D; Goulding, Euginia H; Suganuma, Ryota; Yanagimachi, Ryuzo; Kierszenbaum, Abraham L

    2005-10-01

    Keratin 9 (K9) is one of the components of the perinuclear ring of the manchette found in developing spermatids but is predominantly expressed in the epidermis of the footpad (palm and sole in human epidermis). As an initial step to determine the function of K9 protein in sperm development, we have generated a mutant mouse by homologous recombination of the targeting vector containing the disrupted K9 gene in which the neo(r) gene was inserted into the intron 6. This insertion resulted in the expression of two K9 mRNAs: a wild-type K9 mRNA, in which intron 6 with the neo(r) gene was completely spliced out, and a mutated form in which only a portion of the intron 6 between neo(r) gene and exon 7 was spliced out. While both heterozygous (K9(+/neo)) and homozygous (K9(neo/neo)) mutant mice expressed the wild-type form of K9 protein, the expression profile of the wild-type K9 in K9(neo/neo) mutants was modified. In addition, the open reading frame of the aberrant mRNA terminated at the exon 6/intron 6 splice site, resulting in a truncated K9 protein. Both K9(+neo) and K9(neo/neo) male mice displayed spermatids with ectopic manchette. Coiled tails were seen in maturing spermatids and epididymal sperm of mutant mice and sperm with deformed tails displayed forward motility. A predominant sperm anomaly was residual cytoplasm at the end of the mitochondria-containing middle piece tail segment. The residual cytoplasm displayed vesicles with random in situ motion, suggesting a transport impediment toward the distal end of the sperm tail. All mutant mice were fertile. Surprisingly, in oocyte nuclear injection experiments using K9(neo/neo) sperm donor, 76% of the resulting animals displayed a deletion of the neo(r) gene from the intron 6 of the mutated K9 allele. Results of this study support the view that intron 6 influences the transcriptional efficiency of the K9 gene by decreasing production of wild-type K9 and changing the expression of K9 proteins.

  17. Sorting of Sperm by Morphology

    NASA Astrophysics Data System (ADS)

    Koh, James; Marcos, Marcos

    2016-11-01

    Many studies have proven that the percentage of morphologically normal sperm is a significant factor in determining the success of assisted reproduction. The velocity of sperm in a microchannel with shear flow subjected to an external field will be explored theoretically. The difference in response between morphologically normal and abnormal sperm will be computed from a statistical approach, to study the feasibility and effectiveness of sorting by an external field to remove abnormal sperm. The full name of this author is Marcos.

  18. The Effect of Ambient Air Pollution on Sperm Quality

    PubMed Central

    Hansen, Craig; Luben, Thomas J.; Sacks, Jason D.; Olshan, Andrew; Jeffay, Susan; Strader, Lillian; Perreault, Sally D.

    2010-01-01

    Background Research has suggested an association with ambient air pollution and sperm quality. Objectives We investigated the effect of exposure to ozone (O3) and particulate matter < 2.5 μm in aerodynamic diameter (PM2.5) on sperm quality. Methods We reexamined a previous cohort study of water disinfection by-products to evaluate sperm quality in 228 presumed fertile men with different air pollution profiles. Outcomes included sperm concentration, total sperm per ejaculate (count), and morphology, as well as DNA integrity and chromatin maturity. Exposures to O3 and PM2.5 were evaluated for the 90–day period before sampling. We used multivariable linear regression, which included different levels of adjustment (i.e., without and with season and temperature) to assess the relationship between exposure to air pollutants during key periods of sperm development and adverse sperm outcomes. Results Sperm concentration and count were not associated with exposure to PM2.5, but there was evidence of an association (but not statistically significant) with O3 concentration and decreased sperm concentration and count. Additionally, a significant increase in the percentage of sperm cells with cytoplasmic drop [β = 2.64; 95% confidence interval (CI), 0.21–5.06] and abnormal head (β = 0.47; 95% CI, 0.03–0.92) was associated with PM2.5 concentration in the base model. However, these associations, along with all other sperm outcomes, were not significantly associated with either pollutant after controlling for season and temperature. Overall, although we found both protective and adverse effects, there was generally no consistent pattern of increased abnormal sperm quality with elevated exposure to O3 or PM2.5. Conclusions Exposures to O3 or PM2.5 at levels below the current National Ambient Air Quality Standards were not associated with statistically significant decrements in sperm outcomes in this cohort of fertile men. However, some results suggested effects on sperm

  19. Genetic sperm defects.

    PubMed

    Chenoweth, Peter J

    2005-08-01

    Genetic sperm defects are specific sperm defects, which have been shown to have a genetic mode of transmission. Such genetic linkage, either direct or indirect, has been associated with a number of sperm defects in different species, with this number increasing with improved diagnostic capabilities. A number of sperm defects, which have proven or suspected genetic modes of transmission are discussed herein, with particular emphasis on cattle. These include: 1. Acrosome defects (knobbed, ruffled and incomplete); 2. Head defects (abnormal condensation, decapitated, round head, rolled head, nuclear crest); 3. Midpiece abnormalities ("Dag" defect, "corkscrew" defect, "pseudo-droplet" defect); 4. Tail defects ("tail stump" defect, primary ciliary dyskinesia).

  20. Comprehensive meiotic segregation analysis of a 4-breakpoint t(1;3;6) complex chromosome rearrangement using single sperm array comparative genomic hybridization and FISH.

    PubMed

    Hornak, Miroslav; Vozdova, Miluse; Musilova, Petra; Prinosilova, Petra; Oracova, Eva; Linkova, Vlasta; Vesela, Katerina; Rubes, Jiri

    2014-10-01

    Complex chromosomal rearrangements (CCR) represent rare structural chromosome abnormalities frequently associated with infertility. In this study, meiotic segregation in spermatozoa of an infertile normospermic carrier of a 4-breakpoint t(1;3;6) CCR was analysed. A newly developed array comparative genomic hybridization protocol was used, and all chromosomes in 50 single sperm cells were simultaneously examined. Three-colour FISH was used to analyse chromosome segregation in 1557 other single sperm cells. It was also used to measure an interchromosomal effect; sperm chromatin structure assay was used to measure chromatin integrity. A high-frequency of unbalanced spermatozoa (84%) was observed, mostly arising from the 3:3 symmetrical segregation mode. Array comparative genomic hybridization was used to detect additional aneuploidies in two out of 50 spermatozoa (4%) in chromosomes not involved in the complex chromosome rearrangement. Significantly increased rates of diploidy and XY disomy were found in the CCR carrier compared with the control group (P < 0.001). Defective condensation of sperm chromatin was also found in 22.7% of spermatozoa by sperm chromatin structure assay. The results indicate that the infertility in the man with CCR and normal spermatozoa was caused by a production of chromosomally unbalanced, XY disomic and diploid spermatozoa and spermatozoa with defective chromatin condensation.

  1. Viable offspring obtained from Prm1-deficient sperm in mice

    PubMed Central

    Takeda, Naoki; Yoshinaga, Kazuya; Furushima, Kenryo; Takamune, Kazufumi; Li, Zhenghua; Abe, Shin-ichi; Aizawa, Shin-ichi; Yamamura, Ken-ichi

    2016-01-01

    Protamines are expressed in the spermatid nucleus and allow denser packaging of DNA compared with histones. Disruption of the coding sequence of one allele of either protamine 1 (Prm1) or Prm2 results in failure to produce offspring, although sperm with disrupted Prm1 or Prm2 alleles are produced. Here, we produced Prm1-deficient female chimeric mice carrying Prm1-deficient oocytes. These mice successfully produced Prm1+/− male mice. Healthy Prm1+/− offspring were then produced by transferring blastocysts obtained via in vitro fertilization using zona-free oocytes and sperm from Prm1+/− mice. This result suggests that sperm lacking Prm1 can generate offspring despite being abnormally shaped and having destabilised DNA, decondensed chromatin and a reduction in mitochondrial membrane potential. Nevertheless, these mice showed little derangement of expression profiles. PMID:27250771

  2. Viable offspring obtained from Prm1-deficient sperm in mice.

    PubMed

    Takeda, Naoki; Yoshinaga, Kazuya; Furushima, Kenryo; Takamune, Kazufumi; Li, Zhenghua; Abe, Shin-Ichi; Aizawa, Shin-Ichi; Yamamura, Ken-Ichi

    2016-06-02

    Protamines are expressed in the spermatid nucleus and allow denser packaging of DNA compared with histones. Disruption of the coding sequence of one allele of either protamine 1 (Prm1) or Prm2 results in failure to produce offspring, although sperm with disrupted Prm1 or Prm2 alleles are produced. Here, we produced Prm1-deficient female chimeric mice carrying Prm1-deficient oocytes. These mice successfully produced Prm1(+/-) male mice. Healthy Prm1(+/-) offspring were then produced by transferring blastocysts obtained via in vitro fertilization using zona-free oocytes and sperm from Prm1(+/-) mice. This result suggests that sperm lacking Prm1 can generate offspring despite being abnormally shaped and having destabilised DNA, decondensed chromatin and a reduction in mitochondrial membrane potential. Nevertheless, these mice showed little derangement of expression profiles.

  3. Meiotic abnormalities

    SciTech Connect

    1993-12-31

    Chapter 19, describes meiotic abnormalities. These include nondisjunction of autosomes and sex chromosomes, genetic and environmental causes of nondisjunction, misdivision of the centromere, chromosomally abnormal human sperm, male infertility, parental age, and origin of diploid gametes. 57 refs., 2 figs., 1 tab.

  4. The sperm epigenome and potential implications for the developing embryo.

    PubMed

    Jenkins, Timothy G; Carrell, Douglas T

    2012-06-01

    Recent work in the field of male fertility has yielded significant increases in our understanding of the sperm epigenome and its potential role in embryonic development. These new findings have enabled a broad classification of a normal epigenetic state in the male gamete and have provided insight into the possible etiologies of some idiopathic male infertility cases. Histone retention and modification, protamine incorporation into the chromatin, DNA methylation, and spermatozoal RNA transcripts appear to play important roles in the epigenetic state of mature sperm. These epigenetic factors may reveal a historical record of spermatogenesis, portend future functions in embryogenesis, and help to elucidate mechanism of pluripotency. In contrast to the once held dogma regarding the importance of the paternal epigenome, the unique epigenetic landscape in sperm appears to serve more than the gamete itself and is likely influential in the developing embryo. In fact, growing evidence suggests that mature sperm provide appropriate epigenetic marks that drive specific genes toward activation and contribute to the pluripotent state of the embryonic cells. Although not definitive, the current literature provides evidence for the role of the sperm epigenome in the embryo. Future work must be focused on the characterization of epigenetic abnormalities commonly found in individuals with compromised fertility to further establish this role. Additionally, studies should target the effects of environment and aging on the sperm epigenetic program and subsequent fertility loss to determine the etiology of aberrant epigenetic profiles.

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

    PubMed

    Carretero, M I; Giuliano, S M; Casaretto, C I; Gambarotta, M C; Neild, D M

    2012-05-01

    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.

  6. Low Sperm Count

    MedlinePlus

    ... unknown, it might be related to abnormal testicular temperature regulation. Varicoceles result in reduced quality of the ... can be permanently reduced. Overheating the testicles. Elevated temperatures impair sperm production and function. Although studies are ...

  7. Studies on the role and mode of operation of the very-lysine-rich histones in eukaryote chromatin. The conformation of phi1 histones from marine invertebrate sperm.

    PubMed

    Puigdoménech, P; Cabré, O; Palau, J; Bradbury, E M; Crane-Robinson, C

    1975-11-01

    Proton magnetic resonance, circular dichroism and infrared spectroscopy are used to investigate the secondary and tertiary structure of three very lysine-rich histones from marine invertebrate sperm. At high ionic strength both Arbacia lixula and Holothuria tubulosa histone phi 1 are observed to contain 25-30% alpha-helix, no beta-structure and to form specific folded structures. Both phi 1 proton magnetic resonance spectra have perturbed methyl resonances at chemical shifts close to those observed for calf thymus H1, suggesting analogies in tertiary structure. Mytilus edulis histone phi 1 however, shows no spectroscopic evidence of secondary and tertiary structure on salt addition.

  8. Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status.

    PubMed

    Hamilton, Thais Rose dos Santos; de Castro, Letícia Signori; Delgado, Juliana de Carvalho; de Assis, Patrícia Monken; Siqueira, Adriano Felipe Perez; Mendes, Camilla Mota; Goissis, Marcelo Demarchi; Muiño-Blanco, Teresa; Cebrián-Pérez, José Álvaro; Nichi, Marcílio; Visintin, José Antonio; D'Ávila Assumpção, Mayra Elena Ortiz

    2016-04-01

    Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm.

  9. Developmental abnormalities and changes in cholinesterase activity in sea urchin embryos and larvae from sperm exposed to engineered nanoparticles.

    PubMed

    Gambardella, Chiara; Aluigi, Maria G; Ferrando, Sara; Gallus, Lorenzo; Ramoino, Paola; Gatti, Antonietta M; Rottigni, Marino; Falugi, Carla

    2013-04-15

    The objective of this study is to examine the toxicity of engineered nanoparticles (NPs) that are dispersed in sea water by using an in vivo model. Because many products of nanotechnology contain NPs and are commonly used and well-established in the market, the accidental release of NPs into the air and water is quite possible. Indeed, at the end of their life cycle, some NPs are inevitably released into waste water and can reach marine ecosystem and affect the organisms there. Although there are few data on the presence of NPs in the marine environment, our awareness of their potential impact on environmental and organismal health is growing. Shallow-water benthonic organisms such as sea urchins provide planktonic larvae as a trophic base for finfish juveniles and are exposed to water from estuaries and precipitation. Such organisms can therefore be directly affected by NPs that are dispersed into those media. We evaluated the effects of exposure to different concentrations of nanosilver, titanium oxide and cobalt NPs on the sperm of the sea urchin Paracentrotus lividus by analyzing the functionality and the morphology and biochemistry of the first developmental stages of the sea urchin. Sperm were exposed to sea water containing suspensions of NPs ranging from 0.0001 mg/L to 1 mg/L. Fertilization ability was not affected, but developmental anomalies were identified in embryos from the gastrula to pluteus stages, including morphological alterations of the skeletal rods. In addition, the enzymatic activity (cholinesterase, ChE) of the larvae was measured. Acetylcholinesterase (AChE) and propionylcholinesterase activity (PrChE) was affected in all of the exposed samples. The results did not vary consistently with the concentration of NP, but controls were significantly different from exposed samples. Exposure of sea urchin to these NPs may cause neurotoxic damage, and the altered ChE activity may be involved in skeletogenic aberrations. In conclusion, the sea urchin

  10. Linker DNA destabilizes condensed chromatin.

    PubMed

    Green, G R; Ferlita, R R; Walkenhorst, W F; Poccia, D L

    2001-01-01

    The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.

  11. Influence of the Temperature and the Genotype of the HSP90AA1 Gene over Sperm Chromatin Stability in Manchega Rams

    PubMed Central

    Ramón, Manuel; Salces-Ortiz, Judit; González, Carmen; Pérez-Guzmán, M. Dolores; Garde, J. Julián; García-Álvarez, Olga; Maroto-Morales, Alejandro; Calvo, Jorge H.; Serrano, M. Magdalena

    2014-01-01

    The present study addresses the effect of heat stress on males' reproduction ability. For that, we have evaluated the sperm DNA fragmentation (DFI) by SCSA of ejaculates incubated at 37°C during 0, 24 and 48 hours after its collection, as a way to mimic the temperature circumstances to which spermatozoa will be subject to in the ewe uterus. The effects of temperature and temperature-humidity index (THI) from day 60 prior collection to the date of semen collection on DFI were examined. To better understand the causes determining the sensitivity of spermatozoa to heat, this study was conducted in 60 males with alternative genotypes for the SNP G/C−660 of the HSP90AA1 promoter, which encode for the Hsp90α protein. The Hsp90α protein predominates in the brain and testis, and its role in spermatogenesis has been described in several species. Ridge regression analyses showed that days 29 to 35 and 7 to 14 before sperm collection (bsc) were the most critical regarding the effect of heat stress over DFI values. Mixed model analyses revealed that DFI increases over a threshold of 30°C for maximum temperature and 22 for THI at days 29 to 35 and 7 to 14 bsc only in animals carrying the GG−660 genotype. The period 29–35 bsc coincide with the meiosis I process for which the effect of the Hsp90α has been described in mice. The period 7–14 bsc may correspond with later stages of the meiosis II and early stages of epididymal maturation in which the replacement of histones by protamines occurs. Because of GG−660 genotype has been associated to lower levels of HSP90AA1 expression, suboptimal amounts of HSP90AA1 mRNA in GG−660 animals under heat stress conditions make spermatozoa DNA more susceptible to be fragmented. Thus, selecting against the GG−660 genotype could decrease the DNA fragmentation and spermatozoa thermal susceptibility in the heat season, and its putative subsequent fertility gains. PMID:24465903

  12. Cross species fertilization and development investigated by cat sperm injection into mouse oocytes.

    PubMed

    Xu, Yong-Nan; Cui, Xiang-Shun; Sun, Shao-Chen; Jin, Yong-Xun; Kim, Nam-Hyung

    2011-07-01

    The use of intracytoplasmic sperm injection (ICSI) in model animals is a powerful approach for the study of species-specific fertilization processes and multiploidy embryogenesis. In this study, we examined the fertilization process in mouse oocytes following injection of a single mouse or cat sperm, two mouse spermatozoa or mouse and cat spermatozoa. These treatments did not affect histone H3K9 acetylation or methylation, although the pattern of DNA methylation differed following the injection of cat sperm. Immunocytochemical staining revealed that sperm chromatin was normally incorporated with female mouse chromatin following any of the four injection scenarios. Furthermore, metaphase was successfully entered to reach a normal two-cell stage, and cell division could even persist to produce blastocyst stage embryos. In addition, both mouse and cat Pou5l and Nanog mRNA were expressed in the hybrid embryos. These results suggest that, although epigenetic modification of DNA is affected by the sperm injection treatment, fertilization and cleavage occur in a non-species-specific manner. In addition, despite abnormal division of the chromosomes, intra- and inter-species ICSI produced embryos that could develop into blastocysts.

  13. Mammalian sperm nuclear organization: resiliencies and vulnerabilities.

    PubMed

    Champroux, A; Torres-Carreira, J; Gharagozloo, P; Drevet, J R; Kocer, A

    2016-01-01

    Sperm cells are remarkably complex and highly specialized compared to somatic cells. Their function is to deliver to the oocyte the paternal genomic blueprint along with a pool of proteins and RNAs so a new generation can begin. Reproductive success, including optimal embryonic development and healthy offspring, greatly depends on the integrity of the sperm chromatin structure. It is now well documented that DNA damage in sperm is linked to reproductive failures both in natural and assisted conception (Assisted Reproductive Technologies [ART]). This manuscript reviews recent important findings concerning - the unusual organization of mammalian sperm chromatin and its impact on reproductive success when modified. This review is focused on sperm chromatin damage and their impact on embryonic development and transgenerational inheritance.

  14. Sperm macrocephaly syndrome in a patient without AURKC mutations and with a history of recurrent miscarriage.

    PubMed

    Molinari, Emanuela; Mirabelli, Marzia; Raimondo, Stefania; Brussino, Alessandro; Gennarelli, Gianluca; Bongioanni, Francesca; Revelli, Alberto

    2013-02-01

    This paper reports a case of recurrent miscarriage in a patient affected by a variant phenotype of sperm macrocephaly syndrome (SMS). SMS is usually related to specific sperm characteristics (large head, multiple tail) and homozygous mutations in the aurora kinase C gene (AURKC). However, the present case observed large-headed spermatozoa with no flagellar abnormalities and no mutations detectable by AURKC sequencing. Furthermore, the patient had repeatedly conceived by intracytoplasmic sperm injection, but pregnancy always aborted. This study performed morphological analysis (Papanicolau staining), annexin V/propidium iodide staining, sperm chromatin structure assay (SCSA), fluorescence in-situ hybridization (FISH) and transmission electron microscopy. This study observed large-headed, mono-tailed, mono-centriolar spermatozoa characterized by abnormal chromatin and swollen mitochondria. SCSA revealed a high ratio of late apoptotic cells with fairly intact amount of DNA. The FISH analysis showed 100% disomy rate. As far as is known, this is the first study to include gene sequencing, TEM, cytogenetic analysis and sperm DNA fragmentation in a case of SMS and also to report recurrent miscarriage related to this specific condition. SMS may be associated with important abnormalities of the sperm subcellular structure and with disomy even in the absence of mutations in the AURKC coding sequence. Sperm macrocephaly syndrome (SMS) is a rare condition that affects spermatozoa and is related to infertility. It is characterized by a specific phenotype of large-headed, multi-tailed spermatozoa with an abnormal chromosomal status. A very few pregnancies have been obtained so far in SMS patients by means of IVF procedures. We present a case of SMS that differs from the classical syndrome as we observed large-headed spermatozoa without tail abnormalities. The affected patient had achieved three pregnancies following IVF, but all aborted. We carried out a detailed examination of

  15. Chromatin hydrodynamics.

    PubMed

    Bruinsma, Robijn; Grosberg, Alexander Y; Rabin, Yitzhak; Zidovska, Alexandra

    2014-05-06

    Following recent observations of large scale correlated motion of chromatin inside the nuclei of live differentiated cells, we present a hydrodynamic theory-the two-fluid model-in which the content of a nucleus is described as a chromatin solution with the nucleoplasm playing the role of the solvent and the chromatin fiber that of a solute. This system is subject to both passive thermal fluctuations and active scalar and vector events that are associated with free energy consumption, such as ATP hydrolysis. Scalar events drive the longitudinal viscoelastic modes (where the chromatin fiber moves relative to the solvent) while vector events generate the transverse modes (where the chromatin fiber moves together with the solvent). Using linear response methods, we derive explicit expressions for the response functions that connect the chromatin density and velocity correlation functions to the corresponding correlation functions of the active sources and the complex viscoelastic moduli of the chromatin solution. We then derive general expressions for the flow spectral density of the chromatin velocity field. We use the theory to analyze experimental results recently obtained by one of the present authors and her co-workers. We find that the time dependence of the experimental data for both native and ATP-depleted chromatin can be well-fitted using a simple model-the Maxwell fluid-for the complex modulus, although there is some discrepancy in terms of the wavevector dependence. Thermal fluctuations of ATP-depleted cells are predominantly longitudinal. ATP-active cells exhibit intense transverse long wavelength velocity fluctuations driven by force dipoles. Fluctuations with wavenumbers larger than a few inverse microns are dominated by concentration fluctuations with the same spectrum as thermal fluctuations but with increased intensity.

  16. Sperm and spermatids contain different proteins and bind distinct egg factors.

    PubMed

    Teperek, Marta; Miyamoto, Kei; Simeone, Angela; Feret, Renata; Deery, Michael J; Gurdon, John B; Jullien, Jerome

    2014-09-19

    Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development.

  17. Topology of chromosome centromeres in human sperm nuclei with high levels of DNA damage

    PubMed Central

    Wiland, Ewa; Fraczek, Monika; Olszewska, Marta; Kurpisz, Maciej

    2016-01-01

    Several studies have shown that the ‘poor’ sperm DNA quality appears to be an important factor affecting male reproductive ability. In the case of sperm cells from males with the correct somatic karyotype but with deficient spermatogenesis, resulting in a high degree of sperm DNA fragmentation, we observed changes in the preferential topology of the chromosome 7, 9, 15, 18, X and Y centromeres. The changes occurred in radial localization and may have been directly linked to the sperm chromatin damage. This conclusion is mainly based on a comparison of FISH signals that were observed simultaneously in the TUNEL-positive and TUNEL-negative sperm cells. The analyzed cells originated from the same ejaculated sample and FISH was performed on the same slides, after in situ TUNEL reaction. Based on the observed changes and previous data, it appears that the sperm nucleus architecture can be disrupted by a variety of factors and has a negative influence on spermatogenesis at the same time. Often, these factors coexist (e.g. chromosomal translocations, aneuploidies, a higher DNA fragmentation, abnormal seminology), but no direct correlations between the factors were observed. PMID:27558650

  18. Negative Regulation of p21Waf1/Cip1 by Human INO80 Chromatin Remodeling Complex Is Implicated in Cell Cycle Phase G2/M Arrest and Abnormal Chromosome Stability

    PubMed Central

    Cao, Lingling; Ding, Jian; Dong, Liguo; Zhao, Jiayao; Su, Jiaming; Wang, Lingyao; Sui, Yi; Zhao, Tong; Wang, Fei; Jin, Jingji; Cai, Yong

    2015-01-01

    We previously identified an ATP-dependent human Ino80 (INO80) chromatin remodeling complex which shares a set of core subunits with yeast Ino80 complex. Although research evidence has suggested that INO80 complex functions in gene transcription and genome stability, the precise mechanism remains unclear. Herein, based on gene expression profiles from the INO80 complex-knockdown in HeLa cells, we first demonstrate that INO80 complex negatively regulates the p21Waf1/Cip1 (p21) expression in a p53-mediated mechanism. In chromatin immunoprecipitation (ChIP) and a sequential ChIP (Re-ChIP) assays, we determined that the INO80 complex and p53 can bind to the same promoter region of p21 gene (-2.2kb and -1.0kb upstream of the p21 promoter region), and p53 is required for the recruitment of the INO80 complex to the p21 promoter. RNAi knockdown strategies of INO80 not only led to prolonged progression of cell cycle phase G2/M to G1, but it also resulted in abnormal chromosome stability. Interestingly, high expression of p21 was observed in most morphologically-changed cells, suggesting that negative regulation of p21 by INO80 complex might be implicated in maintaining the cell cycle process and chromosome stability. Together, our findings will provide a theoretical basis to further elucidate the cellular mechanisms of the INO80 complex. PMID:26340092

  19. Negative Regulation of p21Waf1/Cip1 by Human INO80 Chromatin Remodeling Complex Is Implicated in Cell Cycle Phase G2/M Arrest and Abnormal Chromosome Stability.

    PubMed

    Cao, Lingling; Ding, Jian; Dong, Liguo; Zhao, Jiayao; Su, Jiaming; Wang, Lingyao; Sui, Yi; Zhao, Tong; Wang, Fei; Jin, Jingji; Cai, Yong

    2015-01-01

    We previously identified an ATP-dependent human Ino80 (INO80) chromatin remodeling complex which shares a set of core subunits with yeast Ino80 complex. Although research evidence has suggested that INO80 complex functions in gene transcription and genome stability, the precise mechanism remains unclear. Herein, based on gene expression profiles from the INO80 complex-knockdown in HeLa cells, we first demonstrate that INO80 complex negatively regulates the p21Waf1/Cip1 (p21) expression in a p53-mediated mechanism. In chromatin immunoprecipitation (ChIP) and a sequential ChIP (Re-ChIP) assays, we determined that the INO80 complex and p53 can bind to the same promoter region of p21 gene (-2.2 kb and -1.0 kb upstream of the p21 promoter region), and p53 is required for the recruitment of the INO80 complex to the p21 promoter. RNAi knockdown strategies of INO80 not only led to prolonged progression of cell cycle phase G2/M to G1, but it also resulted in abnormal chromosome stability. Interestingly, high expression of p21 was observed in most morphologically-changed cells, suggesting that negative regulation of p21 by INO80 complex might be implicated in maintaining the cell cycle process and chromosome stability. Together, our findings will provide a theoretical basis to further elucidate the cellular mechanisms of the INO80 complex.

  20. Sperm studies in anesthesiologists

    SciTech Connect

    Wyrobek, A.J.; Brodsky, J.; Gordon, l.; Moore, D.H., II; Watchmaker, G.; Cohen, E.N.

    1981-11-01

    Semen samples were collected from 46 anesthesiologists each of whom had worked a minimum of one year in hospital operating rooms ventilated with modern gas-scavenging devices. Samples collected from 26 beginning residents in anesthesiology served as controls. Concentrations of sperm and percentage of sperm having abnormal head shapes were determined for each sample. No significant differences were found between anesthesiologists and beginning residents. Limiting the analyses to men having no confounding factors (varicocele, recent illness, medications, heavy smoking, frequent sauna use) did not change the results. The sperm concentration and morphology in 13 men did not change signficantly after one year of exposure to anesthetic gases. However, the group of men who had one or more confounding factors (excluding exposure to anesthetic gases) showed significantly higher percentages of sperm abnormalities than did the group of men without such factors. These results suggest that limited exposure to anesthetic gases does not significantly affect sperm production as judged by changes in sperm concentration and morphology. These data are reassuring, but since the hospitals surveyed used modern gas-scavenging devices, men who are occupationally exposed to anesthetic gases without this protection should be studied for fuller assessment of the possible human spermatotoxic effects.

  1. Chromatin Dynamics

    PubMed Central

    Hübner, Michael R.; Spector, David L.

    2010-01-01

    The expression patterns of many protein-coding genes are orchestrated in response to exogenous stimuli, as well as cell-type-specific developmental programs. In recent years, researchers have shown that dynamic chromatin movements and interactions in the nucleus play a crucial role in gene regulation. In this review, we highlight our current understanding of the organization of chromatin in the interphase nucleus and the impact of chromatin dynamics on gene expression. We also discuss the current state of knowledge with regard to the localization of active and inactive genes within the three-dimensional nuclear space. Furthermore, we address recent findings that demonstrate the movements of chromosomal regions and genomic loci in association with changes in transcriptional activity. Finally, we discuss the role of intra-and interchromosomal interactions in the control of coregulated genes. PMID:20462379

  2. The relationship of bull fertility to sperm nuclear shape

    USGS Publications Warehouse

    Ostermeier, G.C.; Sargeant, G.A.; Yandell, B.S.; Parrish, J.J.

    2001-01-01

    group had a linear relationship (r .89, P .05) with fertility. To construct a plot of mean sperm shapes, a novel technique to automatically orient and identify the anterior tip of the sperm head was developed. The mean nuclear shape of high-fertility sperm was more elongated and tapered than those of lower fertility. A discriminant function (P .05) was also constructed that separated the 6 bulls into 2 groups based only on the harmonic amplitudes or sperm nuclear shape. The bulls were correctly classified into the 2 fertility groups. A comparison of sperm chromatin structure analysis (SCSA) and harmonic amplitudes found that overall size variance, anterior roundness, and posterior taperedness of sperm nuclei were related to chromatin stability (P .05). Some of the differences observed in sperm nuclear shape between the high- and lower-fertility bulls may be explained by varying levels of chromatin stability. However, sperm nuclear shape appears to contain additional information from chromatin stability alone. In this particular study, with 6 bulls, all with good chromatin quality, sperm nuclear shape was a better predictor of bull fertility.

  3. Patterns of sperm damage in Chernobyl passerine birds suggest a trade-off between sperm length and integrity.

    PubMed

    Hermosell, Ignacio G; Laskemoen, Terje; Rowe, Melissah; Møller, Anders P; Mousseau, Timothy A; Albrecht, Tomás; Lifjeld, Jan T

    2013-10-23

    Interspecific variation in sperm size is enigmatic, but generally assumed to reflect species-specific trade-offs in selection pressures. Among passerine birds, sperm length varies sevenfold, and sperm competition risk seems to drive the evolution of longer sperm. However, little is known about factors favouring short sperm or constraining the evolution of longer sperm. Here, we report a comparative analysis of sperm head abnormalities among 11 species of passerine bird in Chernobyl, presumably resulting from chronic irradiation following the 1986 accident. Frequencies of sperm abnormalities varied between 15.7 and 77.3% among species, more than fourfold higher than in uncontaminated areas. Nonetheless, species ranked similarly in sperm abnormalities in unpolluted areas as in Chernobyl, pointing to intrinsic factors underlying variation in sperm damage among species. Scanning electron microscopy of abnormal spermatozoa revealed patterns of acrosome damage consistent with premature acrosome reaction. Sperm length, but not sperm competition risk explained variation in sperm damage among species. This suggests that longer spermatozoa are more susceptible to premature acrosome reaction. Therefore, we hypothesize a trade-off between sperm length and sperm integrity affecting sperm evolution in passerine birds.

  4. Chromatin remodelling during male gametophyte development.

    PubMed

    Borg, Michael; Berger, Frédéric

    2015-07-01

    The plant life cycle alternates between a diploid sporophytic phase and haploid gametophytic phase, with the latter giving rise to the gametes. Male gametophyte development encompasses two mitotic divisions that results in a simple three-celled structure knows as the pollen grain, in which two sperm cells are encased within a larger vegetative cell. Both cell types exhibit a very different type of chromatin organization - highly condensed in sperm cell nuclei and highly diffuse in the vegetative cell. Distinct classes of histone variants have dynamic and differential expression in the two cell lineages of the male gametophyte. Here we review how the dynamics of histone variants are linked to reprogramming of chromatin activities in the male gametophyte, compaction of the sperm cell genome and zygotic transitions post-fertilization.

  5. In vitro effects of l-carnitine and glutamine on motility, acrosomal abnormality, and plasma membrane integrity of rabbit sperm during liquid-storage.

    PubMed

    Sarıözkan, Serpil; Ozdamar, Saim; Türk, Gaffari; Cantürk, Fazile; Yay, Arzu

    2014-06-01

    This study was designed to evaluate the in vitro effects of l-carnitine and glutamine (Gln) on the sperm quality parameters of liquid-stored rabbit semen maintained up to 24 h at 5°C. Pooled and extended ejaculates were divided into two equal portions. l-Carnitine doses of 0.5, 1 and 2mM were added to the first portion, and glutamine was added at the same doses to the second portion. All samples were cooled to 5°C and examined at 0, 6, 12 and 24 h of liquid storage. Supplementation of the semen extender with three different doses of l-carnitine provided significant increases in the percentage of motile sperm at 12 h (P<0.01), and 24h (P<0.001) and enabled significant protection of the sperm plasma membrane (P<0.01) at 12 and 24h of cool-storage, in comparison to the control samples. Only the 2mM dose of l-carnitine significantly (P<0.01) decreased the rate of acrosomal damage when compared to the control group. Furthermore, all doses of Gln caused a significant (P<0.01) decrease in acrosomal damage at 6h, and provided significant improvement (P<0.01) in sperm motility, acrosomal and plasma membrane integrities at 12 and 24h of liquid storage, when compared to the controls. In conclusion, the supplementation of liquid-stored rabbit semen with l-carnitine and Gln provided a protection for sperm against cool storage-induced functional and structural damages.

  6. Sperm cells as vectors in the production of transgenic animals

    SciTech Connect

    Prince, R.M.

    1993-04-28

    Transgenic animals are used in industry and in biomedical research in order to provide in vivo experimental model systems. Sperm cells have been reported used as vectors in the production of transgenic animals before, however no approach has of yet proven to be successful. Fertilizing eggs with genetically modified sperm would be advantageous in that sperm are readily accessible and stable, and eggs can be fertilized by modified sperm cells in vivo. Recent elucidations regarding the unique manner of DNA packaging in sperm chromatin by protamines has provided us with the insight for developing a method of introducing foreign DNA into sperm which is likely to succeed where others have failed. We have developed a method for mimicking the in vivo system of sperm chromatin toroid subunits in vitro, concentrating these toroids, and fluorescent visualization. Our present work concerns development of a method to successfully deliver DNA across the cell membranes and into the nucleus.

  7. Protective effect of alpha glucosyl hesperidin (G-hesperidin) on chronic vanadium induced testicular toxicity and sperm nuclear DNA damage in male Sprague Dawley rats.

    PubMed

    Vijaya Bharathi, B; Jaya Prakash, G; Krishna, K M; Ravi Krishna, C H; Sivanarayana, T; Madan, K; Rama Raju, G A; Annapurna, A

    2015-06-01

    The study was conducted to evaluate the vanadium-induced testicular toxicity and its effect on sperm parameters, sperm nuclear DNA damage and histological alterations in Sprague Dawley rats and to assess the protective effect of G-hesperidin against this damage. Treatment of rats with vanadium at a dose of 1 mg kg bw(-1) for 90 days resulted in significant reduction in serum testosterone levels, sperm count and motility. Further, a parallel increase in abnormal sperm morphology and adverse histopathological changes in testis was also associated with vanadium administration when compared to normal control. Moreover, sperm chromatin dispersion assay revealed that vanadium induces sperm nuclear DNA fragmentation. A marked increase in testicular malondialdehyde levels and decreased activity of antioxidant enzymes such as superoxide dismutase and catalase indicates vanadium-induced oxidative stress. Co-administration of G-hesperidin at a dose of 25 and 50 mg kg bw(-1) significantly attenuated the sperm parameters and histological changes by restoring the antioxidant levels in rat testis. These results suggested that vanadium exposure caused reduced bioavailability of androgens to the tissue and increased free radical formation, thereby causing structural and functional changes in spermatozoa. G-hesperidin exhibited antioxidant effect by protecting the rat testis against vanadium-induced oxidative damage, further ensures antioxidant potential of bioflavonoids.

  8. Nuclear microscopy of sperm cell elemental structure

    NASA Astrophysics Data System (ADS)

    Bench, Graham S.; Balhorn, Rod; Friz, Alexander M.

    1995-05-01

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility.

  9. Nuclear microscopy of sperm cell elemental structure

    SciTech Connect

    Bench, G.S.; Balhorn, R.; Friz, A.M.; Freeman, S.P.H.T.

    1994-09-28

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility.

  10. The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion.

    PubMed

    Love, C C; Kenney, R M

    1998-10-15

    The relationship between fertility and susceptibility of sperm DNA to denaturation was determined in a group of 84 actively breeding, clinically fertile stallions. Susceptibility of DNA to denaturation was determined using the sperm chromatin structure assay (SCSA). The SCSA measures, mean of alpha-t (mean alpha t), standard deviation of alpha-t (SD alpha t), and the COMP of alpha-t (cells outside the main population)] were significantly correlated with the percentage seasonal pregnancy rate (SPR; mean alpha t, r = -0.24, P < or = 0.05; % COMP alpha t, r = -0.27, P < or = 0.05); percentage pregnant per first cycle (FCP; SD alpha t, r = -0.30, P < or = 0.01; % COMP alpha t, r = -0.42, P < or = 0.0001); and the percentage pregnant per cycle (PC; mean alpha t, r = -0.31, P < or = 0.01; SD alpha t, r = -0.32, P < or = 0.01; % COMP alpha t, r = -0.41, P < or = 0.0001). This study describes detectable intrinsic variation in sperm chromatin structure among fertile stallions (SPR, mean = 83%; FCP, mean = 58%; PC, mean = 57%) in an active breeding population (number of mares bred/stallion/year, mean = 37), in the absence of overt reproductive abnormalities and apparent diseases such that an increase in the susceptibility of sperm DNA to denaturation is associated with reduced fertility, both in terms of efficiency of reproduction (FCP and PC) and seasonal pregnancy rate (SPR). Both COMP alpha t and mean alpha t were useful indicators of fertility, with COMP alpha t being the only SCSA value able to identify mean differences between fertility groupings for SPR and FCP, and overall it was the most reliable indicator of fertility in this group of stallions. The SCSA is able to evaluate a compartment of the spermatozoa which is different from that of traditional tests for sperm quality such as motility and morphology.

  11. Histone H3.3 regulates dynamic chromatin states during spermatogenesis

    PubMed Central

    Yuen, Benjamin T. K.; Bush, Kelly M.; Barrilleaux, Bonnie L.; Cotterman, Rebecca; Knoepfler, Paul S.

    2014-01-01

    The histone variant H3.3 is involved in diverse biological processes, including development, transcriptional memory and transcriptional reprogramming, as well as diseases, including most notably malignant brain tumors. Recently, we developed a knockout mouse model for the H3f3b gene, one of two genes encoding H3.3. Here, we show that targeted disruption of H3f3b results in a number of phenotypic abnormalities, including a reduction in H3.3 histone levels, leading to male infertility, as well as abnormal sperm and testes morphology. Additionally, null germ cell populations at specific stages in spermatogenesis, in particular spermatocytes and spermatogonia, exhibited increased rates of apoptosis. Disruption of H3f3b also altered histone post-translational modifications and gene expression in the testes, with the most prominent changes occurring at genes involved in spermatogenesis. Finally, H3f3b null testes also exhibited abnormal germ cell chromatin reorganization and reduced protamine incorporation. Taken together, our studies indicate a major role for H3.3 in spermatogenesis through regulation of chromatin dynamics. PMID:25142466

  12. Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis.

    PubMed

    Aravindan, G R; Bjordahl, J; Jost, L K; Evenson, D P

    1997-10-10

    Susceptibility of mammalian sperm DNA to low pH- or heat-induced denaturation in situ has shown very strong dose-response relationships with animal and human exposure to chemical and physical toxicants and also fertility potential. In this study, 23 human semen samples representing a wide range in percentage (7-86%) of sperm exhibiting abnormally high susceptibility of DNA in situ to denaturation were studied for the integrity of their DNA using alkaline comet assay (single-cell microgel electrophoresis, pH 10.0). The percentage of comets observed for these samples ranged from 5 to 95%; these data correlated strongly with the percentage of sperm with increased DNA denaturability (r = 0.973; P < 0.001). Labeling of 3' ends of nicked DNA sites with 5-bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP) followed by tagging with FITC-BrdUTP monoclonal antibody and flow cytometry also indicated significantly strong correlations of BrdUTP incorporation with both abnormal susceptibility of DNA to denaturation (r = 0.859, P < 0.001) and comet assay (r = 0.812, P < 0.001). The relationship among susceptibility of sperm chromatin to acid denaturation in situ, BrdUTP incorporation, and formation of comets suggests that DNA fragmentation monitored by these assays may have important physiological relevance in terms of sperm quality and fertility potential.

  13. Teaching resources. Chromatin remodeling.

    PubMed

    Lue, Neal F

    2005-07-26

    This Teaching Resource provides lecture notes and slides for a class covering chromatin remodeling mechanisms and is part of the course "Cell Signaling Systems: a Course for Graduate Students." The lecture begins with a discussion of chromatin organization and then proceeds to describe the process of chromatin remodeling through a review of chromatin remodeling complexes and methods used to study their function.

  14. Porcine sperm vitrification I: cryoloops method.

    PubMed

    Arraztoa, C C; Miragaya, M H; Chaves, M G; Trasorras, V L; Gambarotta, M C; Péndola, C H; Neild, D M

    2016-09-29

    The aims of this study were to evaluate porcine sperm vitrification in cryoloops, with and without two different cryoprotectants and assess two warming procedures. Extended (n = 3; r = 4) and raw (n = 5; r = 2) semen was diluted in media without and with cryoprotectants (4% dimethylformamide and 4% glycerol) to a final concentration of 20 × 10(6) spermatozoa ml(-1) and vitrified using the cryoloops method. Two warming procedures were evaluated: rapid method (30 s at 37°C) and an ultra-rapid method (7 s at 75°C, followed by 30 s at 37°C). Total motility (phase contrast), sperm viability (6-carboxifluorescein diacetate and propidium iodide stain), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated before and after vitrification and analysed using Friedman's test. In all media, the only seminal parameters that were maintained after vitrification were chromatin condensation and integrity. Vitrification of porcine spermatozoon using cryoloops, both in the presence or absence of cryoprotectants and independent of the warming procedure used, permits conservation of sperm chromatin condensation and integrity. It would be interesting to further verify this by producing porcine embryos using vitrified spermatozoon with intracytoplasmic sperm injection.

  15. DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa.

    PubMed

    Vernocchi, Valentina; Morselli, Maria Giorgia; Lange Consiglio, Anna; Faustini, Massimo; Luvoni, Gaia Cecilia

    2014-10-15

    Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies.

  16. Leptin Improves Sperm Cryopreservation via Antioxidant Defense

    PubMed Central

    Fontoura, Paula; Mello, Mariana Duque; Gallo-Sá, Paulo; Erthal-Martins, Maria Cecília; Cardoso, Maria Cecília Almeida; Ramos, Cristiane

    2017-01-01

    Background: Leptin and its receptor are present in spermatozoa; however, the role of leptin in sperm function is still controversial. Our present study aimed at demonstrating the effect of cryopreservation on sperm DNA fragmentation (DNAf) and investigating the possible effects of sperm capacitation techniques and leptin in vitro incubation on frozen-thawed sperm DNAf and oxidative stress. Methods: Samples of 45 normospermic men attending for infertility investigation at Vida Centro de Fertilidade, Rio de Janeiro, Brazil, were frozen and thawed with or without capacitation and leptin incubation prior to freezing. Sperm DNA fragmentation was evaluated by Sperm Chromatin Dispersion Assay before and after cryopreservation and oxidative stress parameters were measured by spectrophotometry with and without leptin incubation. Statistical analysis was performed using paired t test to compare DNAf between groups before and after freeze-thaw cycle, to compare groups before and after capacitation and leptin incubation and oxidative measurements before and after leptin incubation. Statistical significance was considered when p≤0.05. Results: Our results revealed a significant post-thaw rise in sperm DNAf compared with fresh samples (p=0.0003). Sperm DNAf was significantly reduced when sperm capacitation was performed before freezing, when compared to those frozen with no previous capacitation (p=0.01). The addition of leptin to capacitated sperm before freezing reduced DNAf (p<0.0001) and enhanced superoxide dismutase (p=0.001) and glutathione peroxidase (p=0.02) antioxidant enzymes activity. Conclusion: The addition of leptin to capacitated sperm can improve sperm DNA quality following cryopreservation, possibly by inducing the activity of certain antioxidant enzymes. PMID:28377896

  17. Human sperm chromosome analysis after subzonal sperm insemination of hamster oocytes

    SciTech Connect

    Cozzi, J.

    1994-09-01

    Sperm microinjection techniques, subzonal sperm insemination (SUZI) and intracytoplasmic sperm injection (ICSI), have achieved a wide spread clinical application for the treatment of male infertility. To date, only one study has focused on sperm karyotypes after microinjection. Martin et al. reported a very high incidence of abnormal human sperm complements after ICSI into hamster oocytes. In the present study, are reported the first human sperm karyotypes after SUZI of hamster oocytes. Spermatozoa from two control donors were treated by calcium ionophore A23187 and injected under the zona of hamster eggs. The microinjected eggs were then cultured for cytogenetic analysis of the pronuclei. Out of 47 analyzed sperm chromosome metaphases, 5 (10.6%) were abnormal, 4 (8.5%) were hypohaploid and 1 (2.1%) had a structural abnormality. The sex ratio was not significantly different from the expected 1:1 ratio. Rates of chromosomal abnormalities in microinjected spermatozoa were similar to those observed in spermatozoa inseminated with zona free eggs, suggesting that SUZI procedure per se does not increase sperm chromosomal abnormalities.

  18. Evaluation of chromatin integrity of motile bovine spermatozoa capacitated in vitro.

    PubMed

    Reckova, Z; Machatkova, M; Rybar, R; Horakova, J; Hulinska, P; Machal, L

    2008-08-01

    The efficiency of in vitro embryo production is highly variable amongst individual sires in cattle. To eliminate that this variability is not caused by sperm chromatin damage caused by separation or capacitacion, chromatin integrity was evaluated. Seventeen of AI bulls with good NRRs but variable embryo production efficiency were used. For each bull, motile spermatozoa were separated on a Percoll gradient, resuspended in IVF-TALP medium and capacitated with or incubated without heparin for 6 h. Samples before and after separation and after 3-h and 6-h capacitacion or incubation were evaluated by the Sperm Chromatin Structure Assay (SCSA) and the proportion of sperm with intact chromatin structure was calculated. Based on changes in the non-DFI-sperm proportion, the sires were categorized as DNA-unstable (DNA-us), DNA-stable (DNA-s) and DNA-most stable (DNA-ms) bulls (n=3, n=5 and n=9, respectively). In DNA-us bulls, separation produced a significant increase of the mean non-DFI-sperm proportion (p sperm proportion in H+ sperm (p sperm proportion (p sperm proportion remained almost unchanged. In DNA-ms bulls, neither separation nor capacitacion had any effect on the mean non-DFI-sperm proportion. It can be concluded that, although separation and capacitacion may produce some changes in sperm chromatin integrity, these are not associated with different in vitro fertility of the bulls involved.

  19. Structural studies of chromatin and chromosomes. Progress report, March 15--September 15, 1997

    SciTech Connect

    Bradbury, E.M.

    1997-11-01

    This study focused on the following: (1) the structure of chromatin and chromosomes by neutron and x-ray scatter and atomic force microscope; (2) the architecture of human sperm and the structure of sperm by atomic force microscopy (AFM); (3) genome-architecture and higher-order structures in human sperm nuclei; and (4) the effects of histone modifications on the structure of nucleosomes by protein DNA crosslinking method.

  20. Low 17beta-estradiol levels in CNR1 knock-out mice affect spermatid chromatin remodeling by interfering with chromatin reorganization.

    PubMed

    Cacciola, Giovanna; Chioccarelli, Teresa; Altucci, Lucia; Ledent, Catherine; Mason, J Ian; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

    2013-06-01

    The type 1-cannabinoid receptor, CNR1, regulates differentiation of spermatids. Indeed, we have recently reported that the genetic inactivation of Cnr1 in mice influenced chromatin remodeling of spermatids, by reducing histone displacement and then sperm chromatin quality indices (chromatin condensation and DNA integrity). Herein, we have studied, at both central and testicular levels, the molecular signals potentially involved in histone displacement. In particular, investigation of the neuroendocrine axis involved in estrogen production demonstrated down-regulation of the axis supporting FSH/estrogen secretion in Cnr1-knockout male mice. Conversely, Cnr1-knockout male mice treated with 17beta-estradiol showed a weak increase of pituitary Fsh-beta subunit mRNA levels and a rescue of sperm chromatin quality indices demonstrating that estrogens, possibly in combination with FSH secretion, play an important role in regulating chromatin remodeling of spermatids.

  1. Global methylation status of sperm DNA in carriers of chromosome structural aberrations

    PubMed Central

    Olszewska, Marta; Barciszewska, Miroslawa Z; Fraczek, Monika; Huleyuk, Nataliya; Chernykh, Vyacheslav B; Zastavna, Danuta; Barciszewski, Jan; Kurpisz, Maciej

    2017-01-01

    Male infertility might be clearly associated with aberrant DNA methylation patterns in human spermatozoa. An association between oxidative stress and the global methylation status of the sperm genome has also been suggested. The aim of the present study was to determine whether the global sperm DNA methylation status was affected in the spermatozoa of carriers of chromosome structural aberrations. The relationships between the 5-methylcytosine (m5C) levels in spermatozoa and chromatin integrity status were evaluated. The study patients comprised male carriers of chromosome structural aberrations with reproductive failure (n = 24), and the controls comprised normozoospermic sperm volunteers (n = 23). The global m5C level was measured using thin-layer chromatography (TLC) and immunofluorescence (IF) techniques. The sperm chromatin integrity was assessed using aniline blue (AB) staining and TUNEL assay. The mean m5C levels were similar between the investigated chromosome structural aberrations carriers (P) and controls (K). However, sperm chromatin integrity tests revealed significantly higher values in chromosomal rearrangement carriers than in controls (P < 0.05). Although the potential relationship between sperm chromatin integrity status and sperm DNA fragmentation and the m5C level juxtaposed in both analyzed groups (P vs K) was represented in a clearly opposite manner, the low chromatin integrity might be associated with the high hypomethylation status of the sperm DNA observed in carriers of chromosome structural aberrations. PMID:26908061

  2. Chromatin enrichment for proteomics

    PubMed Central

    Kustatscher, Georg; Wills, Karen L. H.; Furlan, Cristina; Rappsilber, Juri

    2015-01-01

    During interphase, chromatin hosts fundamental cellular processes, such as gene expression, DNA replication and DNA damage repair. To analyze chromatin on a proteomic scale, we have developed chromatin enrichment for proteomics (ChEP), which is a simple biochemical procedure that enriches interphase chromatin in all its complexity. It enables researchers to take a ‘snapshot’ of chromatin and to isolate and identify even transiently bound factors. In ChEP, cells are fixed with formaldehyde; subsequently, DNA together with all cross-linked proteins is isolated by centrifugation under denaturing conditions. This approach enables the analysis of global chromatin composition and its changes, which is in contrast with existing chromatin enrichment procedures, which either focus on specific chromatin loci (e.g., affinity purification) or are limited in specificity, such as the analysis of the chromatin pellet (i.e., analysis of all insoluble nuclear material). ChEP takes half a day to complete and requires no specialized laboratory skills or equipment. ChEP enables the characterization of chromatin response to drug treatment or physiological processes. Beyond proteomics, ChEP may preclear chromatin for chromatin immunoprecipitation (ChIP) analyses. PMID:25101823

  3. Sperm Competition, Sperm Numbers and Sperm Quality in Muroid Rodents

    PubMed Central

    Gómez Montoto, Laura; Magaña, Concepción; Tourmente, Maximiliano; Martín-Coello, Juan; Crespo, Cristina; Luque-Larena, Juan José

    2011-01-01

    Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An “overall sperm quality” parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and

  4. Sperm characterization and identification of sperm sub-populations in ejaculates from pampas deer (Ozotoceros bezoarticus).

    PubMed

    Beracochea, F; Gil, J; Sestelo, A; Garde, J J; Santiago-Moreno, J; Fumagalli, F; Ungerfeld, R

    2014-10-01

    Pampas deer (Ozotoceros bezoarticus) is a native endangered species. Knowledge of the basic spermiogram characteristics and the morphometric descriptors is necessary to effectively develop sperm cryopreservation. In other species, sperm sub-population is related to sperm cryo-resistance. The objective was to provide a general description of the sperm, including sperm head morphometric descriptors, its repeatability, and the existence of sperm sub-populations. Sperm were obtained from adult males by electroejaculation during the breeding season. The motility score was 3.4 ± 0.2 (mean ± SEM) and progressive motility was 59.4 ± 3.7%. Ejaculated volume was 413.9 ± 51.0 μl, the total number of sperm ejaculated was 321.2 ± 55.4 × 10(6). Also, 63.3 ± 3.1% of the sperm were morphologically abnormal and 23.7 ± 2.3% had acrosome damage. The sperm head length was 7.6 ± 0.01 μm, width 4.4 ± 0.01 μm, area 28.1 ± 0.07 μm(2) and the perimeter was 21.9 ± 0.04 μm. There was a positive relationship among morphometric descriptors and the motility score, overall motility and progressive motility. Also length (P=0.011), width (P=0.003), area (P=0.006) and perimeter (P=0.009) of sperm head obtained in two different collections were positively related. Overall, the low concentration, volume, overall quality and abnormal morphology, and wide variation of these variables may be a limitation for the development of sperm cryopreserved banks. There were three sperm sub-populations with different morphometric characteristics. The morphometric descriptors are maintained similarly among different collections.

  5. Methods of sperm DNA extraction for genetic and epigenetic studies.

    PubMed

    Griffin, Jeanine

    2013-01-01

    High quality DNA extractions developed for mammalian somatic cells are ineffective for sperm, due mainly to the high degree of nuclear compaction in sperm. The highly specialized nuclear proteins in sperm create a chromatin structure that is at least six times denser than histone bound DNA. Unlike somatic cells, sperm DNA is highly compacted by the replacement of histones with sperm-specific low molecular weight proteins called protamines. Both the protamines and the disulfide bridges formed within and between protamines inhibit the extraction of sperm DNA by standard techniques used for somatic cells. Here we describe the guanidine thiocyanate method reported by Hossain with additional modifications resulting in high molecular weight DNA of high quality with an A260/280 ratio ranging between 1.8 and 2.0 and an A260/230 ratio of 2.0 and greater. The DNA is efficiently digested with restriction enzymes and amplified by PCR.

  6. A model for the control of DNA integrity by the sperm nuclear matrix

    PubMed Central

    Gawecka, Joanna E; Ribas-Maynou, Jordi; Benet, Jordi; Ward, W Steven

    2015-01-01

    The highly condensed chromatin of mammalian spermatozoa is usually considered to be biologically inert before fertilization. However, we have demonstrated that even in this compacted state, sperm chromatin is subject to degradation at open configurations associated with the nuclear matrix through a process we have termed sperm chromatin fragmentation (SCF). This suggests that a mechanism exists to monitor the health of spermatozoa during transit through the male reproductive tract and to destroy the genome of defective sperm cells. The site of DNA damage in SCF, the matrix attachment sites, are the same that we hypothesize initiate DNA synthesis in the zygote. When sperm that have damaged DNA are injected into the oocyte, the newly created zygote responds by delaying DNA synthesis in the male pronucleus and, if the damage is severe enough, arresting the embryo's development. Here we present a model for paternal DNA regulation by the nuclear matrix that begins during sperm maturation and continues through early embryonic development. PMID:25926613

  7. Cytometry of deoxyribonuclei acid content and morphology of mammalian sperm

    SciTech Connect

    Gledhill, B.L.

    1983-01-01

    Because spermatogenesis is exquisitely sensitive to external influences, sperm can serve as a biological dosimeter. Advances in interpreting induced sperm abnormalities require a better understanding of sperm characteristics. This report reviews the application of several methods for automated, quantitative detection of shape changes, methods that are faster and more sensitive than conventional subjective technqiues. Variability of sperm deoxyribonucleic acid content as a bioassay of genetic damage is explored, and limitations of the bioassay are discussed. New flow cytometric techniques that could lead to sexing mammalian sperm are examined.

  8. Cytogenetics of human sperm: Structural aberrations and DNA replication

    SciTech Connect

    Brandriff, B.F.; Gordon, L.A.; Carrano, A.V.

    1989-07-11

    The human sperm-hamster egg system, first introduced in 1978 (Rudak et al), has yielded some important insights into questions on chromosomal integrity of human sperm. In this system, human sperm are co-incubated with eggs from the golden hamster. After the gametes fuse, eggs are cultured overnight and approximately 15 hours after fusion, display the haploid chromosomal complement of individual human sperm cells. These chromosomes can be analyzed by standard banding techniques to identify and quantify structural and numerical abnormalities in single sperm. 32 refs., 1 fig.

  9. Cofilin is correlated with sperm quality and influences sperm fertilizing capacity in humans.

    PubMed

    Chen, S M; Chen, X M; Lu, Y L; Liu, B; Jiang, M; Ma, Y X

    2016-11-01

    Spermatozoa should undergo a series of biochemical modifications in female reproduction tract, which is collectively called sperm capacitation. The capacitated spermatozoa can bind to the egg zona pellucida, resulting in the occurrence of acrosome reaction which enabled spermatozoa penetrate into the egg. The formation of actin plays an important role in these processes. Actin polymerized during sperm capacitation, but the polymers dispersed before acrosome reaction. In this study, we take our focus on actin-binding protein, cofilin. Our results showed that the % and intensity of sperm expressing cofilin in normal sperm were significantly higher than in abnormal sperm, and the sperm expressing cofilin was correlated with sperm quality. Furthermore, treatment with anti-cofilin antibody increased the percentage of sperm capacitation and inhibited progesterone- or A23187- induced acrosome reaction in a dose-dependent manner. The presence of 100 ng/mL anti-cofilin antibodies markedly blocked the sperm penetration of zona-free hamster eggs. Besides, immunofluorescence results revealed that cofilin was colocalized with F-actin in the midpiece of spermatozoa; however, phospho-cofilin was expressed in the tail rather than in the midpiece of spermatozoa, which was not colocalized with F-actin in spermatozoa. Moreover, western blot revealed that phospho-cofilin increased in sperm capacitation, and the total cofilin and cofilin in insoluble fraction increased in acrosome reaction; immunofluorescence results showed that the amount of cofilin in acrosome increased in sperm capacitation. In conclusion, our study revealed that cofilin expression in human sperm is correlated with sperm quality and the alterations of cofilin and phospho-cofilin in fertilization affects sperm capacitation, acrosome reaction, and spermatozoa-oocyte fusion.

  10. ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications.

    PubMed

    Ramalho-Santos, J; Sutovsky, P; Simerly, C; Oko, R; Wessel, G M; Hewitson, L; Schatten, G

    2000-12-01

    We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mulatta). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle.

  11. The nuclear DNA longevity in cryopreserved boar spermatozoa assessed using the Sperm-Sus-Halomax.

    PubMed

    Alkmin, Diego V; Martinez-Alborcia, Maria J; Parrilla, Inmaculada; Vazquez, Juan M; Martinez, Emilio A; Roca, Jordi

    2013-06-01

    The aim of this experimental study was to evaluate the dynamics of nuclear DNA fragmentation in frozen-thawed (FT) boar spermatozoa incubated over time. Using the Sperm Chromatin Dispersion test (Sperm-Sus-Halomax), this study focused special attention on resolving the hypothesis that the original halo shapes around the sperm head could show dynamic changes over the postthawing incubation time. Twenty FT sperm samples from five boars (four per boar) were incubated at 37 °C during 168 hours and sperm motility (assessed using computer-assisted sperm analysis), viability (evaluated using the LIVE/DEAD Sperm Viability Kit), and nuclear DNA fragmentation were analyzed at 0, 0.5, 2, 4, 6, 24, 48, 72, and 168 hours. The percentages of motile and viable spermatozoa progressively decreased during incubation, with no motile and viable spermatozoa less than 10% in all boars at 24 hours of incubation. Four different halo shapes around the sperm head were considered in the Sperm Chromatin Dispersion test: normal, small, large scattered (typical fragmented nuclear DNA), and absent halo, all of them coexisting at the same time in the boar FT semen samples. Sperm with a large scattered halo did not change during postthaw, consistently showing percentages less than 5% over time in all boars. In contrast, the other three sperm populations showed a dynamic evolution over incubation time, characterized by a gradual reduction of sperm with normal halo, proportional to the increment in the sperm showing a small halo, followed by a switch between the sperm with a small halo and sperm with no halo. These results suggest that three of these four sperm populations, those showing small, large scattered, and absent halo, represent spermatozoa with different degrees of nuclear DNA damage, which should be taken into consideration to indicate the percentage of sperm with fragmented nuclear DNA in boar FT semen samples.

  12. Long range chromatin organization

    PubMed Central

    Acuña, Luciana I Gómez; Kornblihtt, Alberto R

    2014-01-01

    Splicing is a predominantly co-transcriptional process that has been shown to be tightly coupled to transcription. Chromatin structure is a key factor that mediates this functional coupling. In light of recent evidence that shows the importance of higher order chromatin organization in the coordination and regulation of gene expression, we discuss here the possible roles of long-range chromatin organization in splicing and alternative splicing regulation. PMID:25764333

  13. A comparative study of spermatozoal chromatin using acridine orange staining and flow cytometry.

    PubMed

    Lewin, L M; Golan, R; Freidlin, P; Shochat, L

    1999-10-01

    Spermatozoa obtained from fish (Clarias gariepinus), human (Homo sapiens), turkeys (Meleagris gallapova), rats (Rattus norvegicus), hamsters (Mesocricetus auratus), and monkeys (Macaca fascicularis) were stained with acridine orange before measuring fluorescence by flow cytometry. These mature sperm from various species produced different intensities of fluorescence while displaying similar ratios of red/green fluorescence. Comparison of the green fluorescence values for the various species showed the sequence (descending order of fluorescence values) human, turkey, monkey, hamster, rat and fish. The DNA complement (as base pairs in the haploid genome) of the various species did not increase in direct proportion to the fluorescence values. This suggests that the DNA was not equally accessible to the dye in the different species tested. The similarity in ratios of red/green fluorescence suggests that the structure of DNA in the chromatin is similar in the different species but abnormal 'satellite' populations of cells that show higher red/green fluorescence ratios than the parent population have been found in sperm samples from monkeys and from some infertile men. Their high red fluorescence intensities were not caused by RNA because treatment with RNAse did not alter the red fluorescence. It is possible that these cells contain larger amounts of denatured (single stranded) DNA.

  14. Not All Sperm Are Equal: Functional Mitochondria Characterize a Subpopulation of Human Sperm with Better Fertilization Potential

    PubMed Central

    Sousa, Ana Paula; Amaral, Alexandra; Baptista, Marta; Tavares, Renata; Caballero Campo, Pedro; Caballero Peregrín, Pedro; Freitas, Albertina; Paiva, Artur; Almeida-Santos, Teresa; Ramalho-Santos, João

    2011-01-01

    Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrial-sensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality. PMID:21448461

  15. Alterations in chromatin structure during early sea urchin embryogenesis.

    PubMed Central

    Savić, A; Richman, P; Williamson, P; Poccia, D

    1981-01-01

    Sea urchin sperm before fertilization possess the longest nucleosome repeat length yet determined for any chromatin. By the time the fertilized egg gives rise to a blastula or gastrula embryo, the chromatin has a considerably shorter repeat length and, in addition, a sequence of different histone variants of H1, H2A, and H2B has appeared. We have investigated the relationship between these variations in histone composition and concomitant alterations in chromatin structure during the earliest stages of embryogenesis in two species of sea urchin. In contrast to the long repeat distance in sperm, chromatin loaded with cleavage stage histones has a much smaller repeat. Later stages containing predominantly alpha histones display an intermediate spacing. More detailed analysis of the events in the first cell cycle was carried out with polyspermically fertilized eggs. During the first 30 min after fertilization, in which sperm-specific H1 is completely replaced by cleavage-stage H1, the male pronuclear repeat remains unchanged. The decrease toward the repeat length of cleavage stages begins at about the time of DNA synthesis. Higher degrees of polyspermy extend the length of the cell cycle, including the duration of S phase and the length of time to reach the first chromosome condensation. At these higher degrees of polyspermy, the decrease in repeat length is also slowed. We conclude that the adjustment of the arrangement of nucleosomes in embryonic chromatin from that found in sperm can occur within the first cell cycle and that its timing is cell-cycle dependent. The adjustment is separable from a corresponding change in H1 composition. Images PMID:6943576

  16. Structural differences in the chromatin from compartmentalized cells of the sea urchin embryo: differential nuclease accessibility of micromere chromatin.

    PubMed Central

    Cognetti, G; Shaw, B R

    1981-01-01

    The chromatin structure of three cell types isolated from the 16-cell stage sea urchin embryo has been probed with micrococcal nuclease. In micromeres, the four small cells at the vegetal pole, the chromatin is found to be considerably more resistant to degradation by micrococcal nuclease than chromatin in the larger mesomere and macromere cells which undergo more cellular divisions and are committed to different developmental fates. The micromeres show an order of magnitude decrease in the initial digestion rate and a limit digest value which is one third that of the larger blastomeres; both observations are suggestive of the formation of a more condensed chromatin structure during the process of commitment, or as the rate of cell division decreases. The decreased sensitivity to nuclease for micromeres is similar to results reported for sperm and larval stages of development. Images PMID:7312627

  17. Changes in Levels of Seminal Nitric Oxide Synthase, Macrophage Migration Inhibitory Factor, Sperm DNA Integrity and Caspase-3 in Fertile Men after Scrotal Heat Stress

    PubMed Central

    Shi, Zhi-Da; Wang, Lei-Guang; Qiu, Yi

    2015-01-01

    Background This study observes changes in levels of seminal nitric oxide (NO), nitric oxide synthase (NOS), macrophage migration inhibitory factor (MIF), sperm DNA integrity, chromatin condensation and Caspase-3in adult healthy men after scrotal heat stress (SHS). Methods Exposure of the scrotum of 25 healthy male volunteers locally at 40–43°C SHS belt warming 40 min each day for successive 2 d per week. The course of SHS was continuously 3 months. Routine semen analysis, hypo-osmotic swelling (HOS) test, Aniline blue (AB) staining, HOS/AB and terminal deoxynucleotidyl transferase-mediated d UDP nick-end labeling (TUNEL) were carried out before, during and after SHS. Seminal NO and NOS contents were determined by nitrate reduction method. The activated Caspase-3 levels of spermatozoa and MIF in seminal plasma were measured by the enzyme-linked immunosorbent assay (ELISA) method. Statistical significance between mean values was determined using statistical ANOVA tests. Results The mean parameters of sperm concentration, motile and progressive motile sperm and normal morphological sperm were significantly decreased in groups during SHS 1, 2 and 3 months compared with those in groups of pre-SHS (P<0.001). Statistically significant differences of sperm DNA fragmentation, normal sperm membrane, and Caspase-3 activity as well as the level of NO, NOS and MIF in semen were observed between the groups before SHS and after SHS 3 months and the groups during SHS 1, 2 and 3 months (P<0.001). After three months of the SHS, various parameters recovered to the level before SHS. WBC in semen showed a positively significant correlation with the levels of NO, NOS, MIF and Caspase-3 activity. The percentage of abnormal sperm by using the test of HOS showed a positively significant correlation with that of HOS/AB. Conclusions The continuously constant SHS can impact the semen quality and sperm DNA and chromatin, which may be contributed to the high level of NO, NOS, MIF and Caspase

  18. The Effects of Different Doses of Ketamine on Quality of Normal Ejaculated Sperm

    PubMed Central

    Absalan, Forouzan; Ghannadi, Alireza; Zabihi, Abdollah

    2014-01-01

    Background Ketamine, an injectable anesthetic in human and animal medicine, is also a recreational drug used by young adults. The aim of this study is to evaluate the effects of ketamine on membrane integrity, DNA fragmentation and sperm parameters in humans. Materials and Methods This prospective study was conducted on 40 males with normal semen samples over one month (August 2012). Subjects were randomly allocated to four groups (Control and case I, II and III) whose semen samples were adjusted to different concentrations of ketamine (1, 3, 5 µL) for one hour. Sperm analysis was performed for routine parameters, motility and morphology. Evaluation of membrane integrity and DNA fragmentation was done by eosin-Y staining and the sperm chromatin dispersion (SCD) test, respectively. The results were analyzed by ANOVA and Tukey’s tests. P≤0.05 was considered statistically significant. Results Total sperm motility in all case groups were significantly lower compared with the control group. In case group III, progressive motility showed significant difference with case group II. After addition of ketamine, sperm had evidence of coiled tails in all case groups compared to the control group however this observation was not significant. Evaluation of membrane integrity showed the rate of necrospermia increased in all case groups. However, ketamine only significantly affected membrane integrity in case group III. SCD staining showed that in the control group nucleoids with medium halos (63.44 ± 1.2) were significantly different compared to the case groups I (15.44 ± 0.45), II (9.05±1.16) and III (10.55 ± 1.14), respectively. Between case groups, nucleoids with large and medium halos showed significant differences in case groups II and III compared with case group I. Nucleoids with medium halos were significantly different between case groups II and III. Conclusion Ketamine, through its effect on membrane integrity and DNA fragmentation, decreased sperm viability

  19. In vitro effects of nicotine on sperm motility and bio-functional flow cytometry sperm parameters.

    PubMed

    Condorelli, R A; La Vignera, S; Giacone, F; Iacoviello, L; Vicari, E; Mongioi', L; Calogero, A E

    2013-01-01

    The aim of this experimental study was to evaluate the effects of nicotine on sperm motility and on non-conventional sperm parameters in vitro. Capacitated spermatozoa isolated from 10 normozoospermic, healthy, non-smoker men were evaluated. Spermatozoa were exposed to increasing concentrations of nicotine (0, 1, 10, and 100 ng/ml) for 3 and 24 hours. Progressive motility and the following non-conventional sperm parameters, evaluated by flow cytometry, were assessed: mitochondrial membrane potential, viability, phosphatidylserine externalization, late apoptosis, degree of chromatin compactness, and DNA fragmentation. Nicotine suppressed, in a concentration-dependent manner, sperm progressive motility starting from the lowest concentration used (1 ng/ml). Similarly, it reduced the percentage of viable spermatozoa and increased the number of spermatozoa in late apoptosis, with altered chromatin compactness, or DNA fragmentation already after 3 hours of incubation. These effects were observed at a concentration similar (100 ng/ml) to that found in the seminal plasma of smokers (70 ng/ml), with the exception of the effects on sperm DNA fragmentation whose significant effect was detected also at a lower concentration (10 ng/ml). Nicotine may be regarded as a noxious component of cigarette smoke on the male reproductive function.

  20. Sperm DNA oxidative damage and DNA adducts

    PubMed Central

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-01-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps = 0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps = 0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps = 0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on

  1. Sperm is epigenetically programmed to regulate gene transcription in embryos

    PubMed Central

    Teperek, Marta; Simeone, Angela; Gaggioli, Vincent; Miyamoto, Kei; Allen, George E.; Erkek, Serap; Kwon, Taejoon; Marcotte, Edward M.; Zegerman, Philip; Bradshaw, Charles R.; Peters, Antoine H.F.M.; Gurdon, John B.; Jullien, Jerome

    2016-01-01

    For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health. PMID:27034506

  2. Fluorescence In-Situ Hybridization Detects Increased Sperm Aneuploidy in Men with Recurrent Pregnancy Loss

    PubMed Central

    Ramasamy, Ranjith; Scovell, Jason M.; Kovac, Jason R.; Cook, Peter J.; Lamb, Dolores J.; Lipshultz, Larry I.

    2015-01-01

    Objective To investigate, in men presenting with recurrent pregnancy loss (RPL), the prevalence of sperm autosome and sex chromosome aneuploidy. Design Retrospective Study. Setting Male infertility clinic at a tertiary referral center. Patients 140 men with recurrent pregnancy loss provided semen samples and five normozoospermic controls provided 140 semen samples for comparison. RPL, documented in the female partners, was defined as a prior miscarriage and/or recurrent IVF/ICSI failure. Interventions Fluorescent In situ hybridization (FISH) was used to detect numerical abnormalities in sex chromosomes (X,Y) and autosomes (13, 18, 21) in ejaculated sperm. Main Outcome Measures Sperm aneuploidy in men with RPL and normozoospermic controls. Results Men with RPL had a greater percentage of sperm aneuploidy within the sex chromosomes, chromosomes 18 and 13/21 (1.04% vs. 0.38%; 0.18% vs. 0.03%; 0.26% vs. 0.08%). In total, 40% of men with normal sperm density and motility had abnormal sperm aneuploidy in the all the chromosomes analyzed. Men with abnormal sperm density and motility had a higher proportion of sperm sex chromosome aneuploidy than men with normal density/motility (62% vs. 45%). Men with normal strict morphology (>4%) had lower rates of sex chromosome and sperm aneuploidy than men with abnormal strict morphology (28% vs. 57%). There was no association between sperm DNA fragmentation and sperm aneuploidy. Conclusions Men with RPL have increased sperm aneuploidy compared to controls. A total of 40% of men with RPL and normal sperm density/motility had abnormal sperm aneuploidy. Men with oligoasthenozoospermia and abnormal strict morphology had greater percentage of sperm aneuploidy compared to men with normal semen parameters. PMID:25707335

  3. Neutron scatter studies of chromatin structures related to functions

    SciTech Connect

    Bradbury, E.M.

    1992-01-01

    We have made considerable progress in chromatin reconstitution with very lysine rich histone H1/H5 and in understanding the dynamics of nucleosomes. A ferromagnetic fluid was developed to align biological molecules for structural studies using small-angle-neutron-scattering. We have also identified and characterized in intrinsically bent DNA region flaking the RNA polymerase I binding site of the ribosomal RNA gene in Physarum Polycephalum. Finally projects in progress are in the areas of studying the interactions of histone H4 amino-terminus peptide 1-23 and acetylated 1-23 peptide with DNA using thermal denaturation; study of GGAAT repeats found in human centromeres using high resolution Nuclear Magnetic Resonance and nuclease sentivity assay; and the role of histones and other sperm specific proteins with sperm chromatin.

  4. Neutron scatter studies of chromatin structures related to functions

    SciTech Connect

    Bradbury, E.M.

    1992-01-01

    Despite of setbacks in the lack of neutrons for the proposed We have made considerable progress in chromatin reconstitution with the VLR histone H1/H5 and in understanding the dynamics of nucleosomes. A ferromagnetic fluid was developed to align biological molecules for structural studies using small-angle-neutron-scattering. We have also identified and characterized an intrinsically bent DNA region flanking the RNA polymerase I binding site of the ribosomal RNA gene in Physarum Polycephalum. Finally projects in progress are in the areas of studying the interatctions of histone H4 amino-terminus peptide 1-23 and acetylated 1-23 peptide with DNA using thermal denaturation; study of GGAAT repeats found in human centromeres using high resolution Nuclear magnetic Resonance and nuclease sentivity assay; and the role of histones and other sperm specific proteins with sperm chromatin.

  5. Chromatin Topological Transitions

    NASA Astrophysics Data System (ADS)

    Lavelle, C.; Bancaud, A.; Recouvreux, P.; Barbi, M.; Victor, J.; Viovy, J.

    DNA transaction events occurring during a cell cycle (transcription,repair, replication) are always associated with severe topological constraints on the double helix. However, since nuclear DNA is bound to various proteins (including histones) that control its accessibility and 3D organization, these topological constraints propagate or accumulate on a chromatin substrate. This paper focuses on chromatin fiber response to physiological mechanical constraints expected to occur during transcription elongation. We will show in particular how recent single molecule techniques help us to understand how chromatin conformational dynamics could manage harsh DNA supercoiling changes.

  6. Advances in understanding paternally transmitted Chromosomal Abnormalities

    SciTech Connect

    Marchetti, F; Sloter, E; Wyrobek, A J

    2001-03-01

    Multicolor FISH has been adapted for detecting the major types of chromosomal abnormalities in human sperm including aneuploidies for clinically-relevant chromosomes, chromosomal aberrations including breaks and rearrangements, and other numerical abnormalities. The various sperm FISH assays have been used to evaluate healthy men, men of advanced age, and men who have received mutagenic cancer therapy. The mouse has also been used as a model to investigate the mechanism of paternally transmitted genetic damage. Sperm FISH for the mouse has been used to detect chromosomally abnormal mouse sperm, while the PAINT/DAPI analysis of mouse zygotes has been used to evaluate the types of chromosomal defects that can be paternally transmitted to the embryo and their effects on embryonic development.

  7. Effects of toxic work environments on sperm quality and ascorbic acid levels

    SciTech Connect

    Dawson, E.B.; Harris, W.A.; Powell, L.C. )

    1990-02-26

    Surveys have shown that toxic work environments lower sperm quality, and controlled studies indicate that ascorbic acid supplementation improves sperm viability and agglutination. The sperm quality of 50 subjects each from: (1) office workers, (2) a lead smelter, (3) petroleum refineries, and (4) a herbicide plant were compared with serum and semen ascorbic acid levels. The sperm characteristics studied were: count as million/ml and as percent; viability, motility, clumping, and abnormal morphology. The serum ascorbic acid levels were directly proportional to sperm viability and inversely correlated to clumping of all groups. Moreover, serum ascorbic acid levels were also inversely correlated to twin tail and amorphous forms of abnormal sperm morphology. The results of the study indicate that toxic environments depress sperm quality and suggest that ascorbic acid supplementation will improve sperm quality and fertility.

  8. X-ray laser microscopy of rat sperm nuclei.

    PubMed

    Da Silva, L B; Trebes, J E; Balhorn, R; Mrowka, S; Anderson, E; Attwood, D T; Barbee, T W; Brase, J; Corzett, M; Gray, J

    1992-10-09

    The development of high brightness and short pulse width (< 200 picoseconds) x-ray lasers now offers biologists the possibility of high-resolution imaging of specimens in an aqueous environment without the blurring effects associated with natural motions and chemical erosion. As a step toward developing the capabilities of this type of x-ray microscopy, a tantalum x-ray laser at 44.83 angstrom wavelength was used together with an x-ray zone plate lens to image both unlabeled and selectively gold-labeled dried rat sperm nuclei. The observed images show approximately 500 angstrom features, illustrate the importance of x-ray microscopy in determining chemical composition, and provide information about the uniformity of sperm chromatin organization and the extent of sperm chromatin hydration.

  9. X-ray laser microscopy of rat sperm nuclei

    SciTech Connect

    Da Silva, L.B. ); Trebes, J.E.; Balhorn, R.; Mrowka, S.; Barbee, T.W.Jr.; Brase, J.; Corzett, M.; Koch, J.A.; Lee, C.; London, R.A.; MacGowan, B.J.; Matthews, D.L.; Stone, G. ); Anderson, E.; Attwood, D.T. ); Gray, J. ); Kern, D. )

    1992-10-09

    The development of high brightness and short pulse width x-ray lasers now offers biologists the possibility of high-resolution imaging of specimens in an aqueous environment without the blurring effects associated with natural motions and chemical erosion. As a step toward developing the capabilities of this type of x-ray microscopy, a tantalum x-ray laser at 44.83 angstrom wavelength was used together with an x-ray zone plate lens to image both unlabeled and selectively gold-labeled dried rat sperm nuclei. The observed images show {approximately}500 angstrom features, illustrate the importance of x-ray microscopy in determining chemical composition, and provide information about the uniformity of sperm chromatin organization and the extent of sperm chromatin hydration.

  10. Etiologies of sperm oxidative stress

    PubMed Central

    Sabeti, Parvin; Pourmasumi, Soheila; Rahiminia, Tahereh; Akyash, Fatemeh; Talebi, Ali Reza

    2016-01-01

    Sperm is particularly susceptible to reactive oxygen species (ROS) during critical phases of spermiogenesis. However, the level of seminal ROS is restricted by seminal antioxidants which have beneficial effects on sperm parameters and developmental potentials. Mitochondria and sperm plasma membrane are two major sites of ROS generation in sperm cells. Besides, leukocytes including polymer phonuclear (PMN) leukocytes and macrophages produce broad category of molecules including oxygen free radicals, non-radical species and reactive nitrogen species. Physiological role of ROS increase the intracellular cAMP which then activate protein kinase in male reproductive system. This indicates that spermatozoa need small amounts of ROS to acquire the ability of nuclear maturation regulation and condensation to fertilize the oocyte. There is a long list of intrinsic and extrinsic factors which can induce oxidative stress to interact with lipids, proteins and DNA molecules. As a result, we have lipid peroxidation, DNA fragmentation, axonemal damage, denaturation of the enzymes, over generation of superoxide in the mitochondria, lower antioxidant activity and finally abnormal spermatogenesis. If oxidative stress is considered as one of the main cause of DNA damage in the germ cells, then there should be good reason for antioxidant therapy in these conditions. PMID:27351024

  11. Congenital Abnormalities

    MedlinePlus

    ... Listen Español Text Size Email Print Share Congenital Abnormalities Page Content Article Body About 3% to 4% ... of congenital abnormalities earlier. 5 Categories of Congenital Abnormalities Chromosome Abnormalities Chromosomes are structures that carry genetic ...

  12. Comparative profiling of the sperm proteome.

    PubMed

    Holland, Ashling; Ohlendieck, Kay

    2015-02-01

    The highly complex and species-selective mechanism of fertilization is a central theme of developmental biology. Gametogenesis, sperm activation, and egg-sperm recognition are fundamental biological processes, warranting detailed studies into the molecular composition of gametes. Biological MS has been instrumental for the comprehensive itemizing of gamete proteomes. The protein constellation of sperm cells and its subcellular structures has been established for a variety of animal species. Spermatogenesis and the crucial activation of sperm cells as a prerequisite of successful fertilization and physiological adaptations to external stressors was investigated using proteomics, as well as the underlying mechanisms of male infertility with respect to proteome-wide alterations. This review outlines recent achievements of sperm proteomics and exemplifies the usefulness of gel-based surveys by outlining the comparative analysis of abnormal spermatozoa in globozoospermia. Besides label-free MS techniques and cell-based labeling methodology, high-resolution fluorescence 2DE has been shown to be highly suitable as a proteomic biomarker discovery tool in sperm protein research. The appropriateness of novel protein markers for improving our understanding of normal spermatogenesis and sperm activation versus the molecular pathogenesis of male infertility will be discussed. New biomarker candidates might be useful to improve diagnostic, prognostic, and therapeutic aspects of infertility.

  13. Genetic dosage and position effect of small supernumerary marker chromosome (sSMC) in human sperm nuclei in infertile male patient

    PubMed Central

    Olszewska, Marta; Wanowska, Elzbieta; Kishore, Archana; Huleyuk, Nataliya; Georgiadis, Andrew P.; Yatsenko, Alexander N.; Mikula, Mariya; Zastavna, Danuta; Wiland, Ewa; Kurpisz, Maciej

    2015-01-01

    Chromosomes occupy specific distinct areas in the nucleus of the sperm cell that may be altered in males with disrupted spermatogenesis. Here, we present alterations in the positioning of the human chromosomes 15, 18, X and Y between spermatozoa with the small supernumerary marker chromosome (sSMC; sSMC+) and spermatozoa with normal chromosome complement (sSMC−), for the first time described in the same ejaculate of an infertile, phenotypically normal male patient. Using classical and confocal fluorescent microscopy, the nuclear colocalization of chromosomes 15 and sSMC was analyzed. The molecular cytogenetic characteristics of sSMC delineated the karyotype as 47,XY,+der(15)(pter->p11.2::q11.1->q11.2::p11.2->pter)mat. Analysis of meiotic segregation showed a 1:1 ratio of sSMC+ to sSMC− spermatozoa, while evaluation of sperm aneuploidy status indicated an increased level of chromosome 13, 18, 21 and 22 disomy, up to 7 × (2.7 − 15.1). Sperm chromatin integrity assessment did not reveal any increase in deprotamination in the patient’s sperm chromatin. Importantly, we found significant repositioning of chromosomes X and Y towards the nuclear periphery, where both chromosomes were localized in close proximity to the sSMC. This suggests the possible influence of sSMC/XY colocalization on meiotic chromosome division, resulting in abnormal chromosome segregation, and leading to male infertility in the patient. PMID:26616419

  14. Cigarette smoking and its possible effects on sperm

    SciTech Connect

    Kulikauskas, V.; Blaustein, D.; Ablin, R.J.

    1985-10-01

    The possible effects of cigarette smoking on sperm were evaluated by comparison of the quality of sperm from 103 smokers and 135 nonsmokers in a blind study. Smokers were found to possess significantly decreased density (number) and motility of their sperm than nonsmokers. Morphologic abnormalities, particularly bicephalia, although prevalent among individual smokers, did not differ significantly when a comparison of smokers versus nonsmokers was made as a whole. Based on these observations and those of others demonstrating the presence of the mutagenic properties of smoke condensates, the authors suggest that decreases in sperm density and motility in cigarette smokers may be reflective of smoke condensate-induced mutagenic spermatogenital alterations.

  15. Nuclear reprogramming of sperm and somatic nuclei in eggs and oocytes.

    PubMed

    Teperek, Marta; Miyamoto, Kei

    2013-01-01

    Eggs and oocytes have a prominent ability to reprogram sperm nuclei for ensuring embryonic development. The reprogramming activity that eggs/oocytes intrinsically have towards sperm is utilised to reprogram somatic nuclei injected into eggs/oocytes in nuclear transfer (NT) embryos. NT embryos of various species can give rise to cloned animals, demonstrating that eggs/oocytes can confer totipotency even to somatic nuclei. However, many studies indicate that reprogramming of somatic nuclei is not as efficient as that of sperm nuclei. In this review, we explain how and why sperm and somatic nuclei are differentially reprogrammed in eggs/oocytes. Recent studies have shown that sperm chromatin is epigenetically modified to be adequate for early embryonic development, while somatic nuclei do not have such modifications. Moreover, epigenetic memories encoded in sperm chromatin are transgenerationally inherited, implying unique roles of sperm. We also discuss whether somatic nuclei can be artificially modified to acquire sperm-like chromatin states in order to increase the efficiency of nuclear reprogramming.

  16. Dietary supplementation with astaxanthin may ameliorate sperm parameters and DNA integrity in streptozotocin-induced diabetic rats

    PubMed Central

    Bahmanzadeh, Maryam; Vahidinia, Aliasghar; Mehdinejadiani, Shayesteh; Shokri, Saeed

    2016-01-01

    Objective Diabetes mellitus (DM) is known to cause many systemic complications as well as male infertility. Astaxanthin (ASTX) is a powerful antioxidant that is involved in a variety of biologically active processes, including those with anti-diabetes effects. The present study investigates the effect of ASTX on the spermatozoa function in streptozotocin (STZ)-induced diabetic rats. Methods We divided 30 adult rats into three groups (10 rats per group), with a control group that received corn oil mixed with chow. DM was induced by intra-peritoneal injection of STZ. Eight weeks after the STZ injection, half of the diabetic animals were used as diabetic controls, and the rest were treated with ASTX for 56 days. Then the parameters and chromatin integrity of the epididymal sperm were analyzed using chromomycin A3, toluidine blue (TB), and acridine orange (AO) staining. Results The count, viability, and motility of the epididymal sperm were decreased significantly in the STZ group in comparison with the control group (count and viability, p<0.001; motility, p<0.001;0.01). ASTX increased normal morphology and viable spermatozoa compared to the STZ group (morphology, p=0.001; viability, p<0.001;0.05). The percentage of abnormal chromatins in TB and AO staining was higher in the STZ group compared to the control group (p<0.001;0.001). The mean percentage of TB and AO positive spermatozoa in STZ rats was significantly lower in the STZ+ASTX group (TB, p=0.001; AO, p<0.001;0.05). Conclusion This study observed that in vivo ASTX treatment partially attenuates some detrimental effect of diabetes. Conversely, ASTX improved sperm viability, normal morphology, and DNA integrity. PMID:27358826

  17. Functional deficit of sperm and fertility impairment in men with antisperm antibodies.

    PubMed

    Bozhedomov, V A; Nikolaeva, M A; Ushakova, I V; Lipatova, N A; Bozhedomova, G E; Sukhikh, G T

    2015-11-01

    Autoimmune reactions against the sperm cells play an ambiguous role in fertility impairment. The objective of this study was to characterize functional deficit of sperm conditioned by antisperm immune response in normozoospermic men. This was a multi-centric, cross-sectional, сase-control study. The study subjects were 1060 infertile normozoospermic men and 107 fertile men. The main outcome measures were clinical examination, semen analysis including MAR test for antisperm antibodies (ASA), computer-aided sperm analysis, acrosome reaction (AR) detected with flow cytometry, DNA fragmentation measured with sperm chromatin dispersion, reactive oxygen species (ROS) assessed using the luminol-dependent chemiluminescence method. 2% of the fertile men had MAR-IgG ≥ 50%, but all subjects with MAR-IgG>12% were outliers; 16% infertile men had MAR-IgG ≥ 50% (p<0.0001). There was a direct correlation between the infertility duration and MAR-IgG (R=0.3; р<0.0001). The ASA-positive infertile men had AR disorders 2.1 times more frequently (р<0.02), predominantly inductivity disorders. We found signs of hyperactivation proportionate to the ASA level (p<0.001). DNA fragmentation was more highly expressed and was 1.6 and 1.3 times more frequent compared with the fertile and the ASA-negative patients, respectively (p<0.001 and p<0.05). We found signs of oxidative stress (OS): ROS generation by washed ASA-positive spermatozoa was 3.7 times higher than in the fertile men (p<0.00001) and depended on the ASA levels (R=0.5; p<0.0001). The ASA correlation with ROS generation in native sperm was weak (R=0.2; р<0.001). We concluded that autoimmune reactions against spermatozoa are accompanied by a fertility decrease in normozoospermia. This results from AR and capacitation disorders and DNA fragmentation. The pathogenesis of sperm abnormalities in immune infertility is associated with the OS of spermatozoa.

  18. Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation.

    PubMed

    Balasuriya, A; Serhal, P; Doshi, A; Harper, J C

    2014-03-01

    Sperm preparation techniques in assisted reproduction technologies (ART) are potential generators of exogenous stresses that cause additional DNA damage. DNA fragmentation tests, such as the sperm chromatin structure assay, involve freezing sperm samples in the absence of cryoprotectant. Thermal, oxidative stress (OS) and freezing are detrimental to sperm DNA fragmentation and phosphatidylserine (PS) translocation. The primary aim of this study was to subject mature sperm to environmental insults that normally occur during ART. We tested the hypotheses that OS, thermal stress and freeze-thawing caused sperm nuclear and membrane damage and that a positive correlation exists between PS translocation and DNA fragmentation. Sperm DNA integrity deteriorates in semen samples from men with advancing age and a sperm concentration of <15 m ml(-1) . The significant increase in sperm DNA fragmentation at 37 °C after merely 1 h is important clinically as semen liquefaction and short-term sperm storage in an ART cycle involve incubating samples at this temperature. Freezing without a cryoprotectant significantly increases the level of sperm nuclear damage, so it is important not to freeze neat semen prior to DNA fragmentation testing. This study highlights the importance of minimising the production of exogenous stresses during sperm preparation in ART.

  19. Porcine sperm vitrification II: Spheres method.

    PubMed

    Arraztoa, C C; Miragaya, M H; Chaves, M G; Trasorras, V L; Gambarotta, M C; Neild, D M

    2016-11-10

    Owing to current problems in boar sperm cryopreservation, this study proposes to evaluate vitrification in spheres as an alternative cryopreservation procedure, comparing the use or not of permeable cryoprotectants and two warming methods. Extended (n = 3; r = 4) and raw (n = 5; r = 2) porcine spermatozoa were diluted in media, in the absence or presence of either 4% dimethylformamide or 4% glycerol, to a final concentration of 5 × 10(6)  spermatozoa/ml and vitrified using the spheres method. Two warming procedures were evaluated: a rapid method (30 s at 37°C) and an ultrarapid method (7 s at 75°C, followed by 30 s at 37°C). Percentages of total motility (phase contrast), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), sperm viability (6-carboxyfluorescein diacetate and propidium iodide stain), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated in the samples before and after vitrification. Results, analysed using Friedman's test, suggest that rapid warming of raw porcine spermatozoa vitrified without permeable cryoprotectants may preserve DNA condensation and integrity better than the other processing methods studied in this work. Hence, porcine sperm vitrification using spheres could be used to produce embryos with ICSI to further validate this method.

  20. High resolution DNA content measurements of mammalian sperm

    SciTech Connect

    Pinkel, D.; Lake, S.; Gledhill, B.L.; Van Dilla, M.A.; Stephenson, D.; Watchmaker, G.

    1982-01-01

    The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +-7/sup 0/. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative content of these two populations in sperm from normal mice and those with the Cattanach (7 to X) translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.

  1. Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach.

    PubMed

    Lydka, M; Piasecka, M; Gaczarzewicz, D; Koziorowski, M; Bilinska, B

    2012-08-01

    Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 μg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p < 0.05) in flutamide-treated boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.

  2. Higher-order genome organization in platypus and chicken sperm and repositioning of sex chromosomes during mammalian evolution.

    PubMed

    Tsend-Ayush, Enkhjargal; Dodge, Natasha; Mohr, Julia; Casey, Aaron; Himmelbauer, Heinz; Kremitzki, Colin L; Schatzkamer, Kyriena; Graves, Tina; Warren, Wesley C; Grützner, Frank

    2009-02-01

    In mammals, chromosomes occupy defined positions in sperm, whereas previous work in chicken showed random chromosome distribution. Monotremes (platypus and echidnas) are the most basal group of living mammals. They have elongated sperm like chicken and a complex sex chromosome system with homology to chicken sex chromosomes. We used platypus and chicken genomic clones to investigate genome organization in sperm. In chicken sperm, about half of the chromosomes investigated are organized non-randomly, whereas in platypus chromosome organization in sperm is almost entirely non-random. The use of genomic clones allowed us to determine chromosome orientation and chromatin compaction in sperm. We found that in both species chromosomes maintain orientation of chromosomes in sperm independent of random or non-random positioning along the sperm nucleus. The distance of loci correlated with the total length of sperm nuclei, suggesting that chromatin extension depends on sperm elongation. In platypus, most sex chromosomes cluster in the posterior region of the sperm nucleus, presumably the result of postmeiotic association of sex chromosomes. Chicken and platypus autosomes sharing homology with the human X chromosome located centrally in both species suggesting that this is the ancestral position. This suggests that in some therian mammals a more anterior position of the X chromosome has evolved independently.

  3. Higher-order genome organization in platypus and chicken sperm and repositioning of sex chromosomes during mammalian evolution

    PubMed Central

    Tsend-Ayush, Enkhjargal; Dodge, Natasha; Mohr, Julia; Casey, Aaron; Himmelbauer, Heinz; Kremitzki, Colin L.; Schatzkamer, Kyriena; Graves, Tina; Warren, Wesley C.

    2013-01-01

    In mammals, chromosomes occupy defined positions in sperm, whereas previous work in chicken showed random chromosome distribution. Monotremes (platypus and echidnas) are the most basal group of living mammals. They have elongated sperm like chicken and a complex sex chromosome system with homology to chicken sex chromosomes. We used platypus and chicken genomic clones to investigate genome organization in sperm. In chicken sperm, about half of the chromosomes investigated are organized non-randomly, whereas in platypus chromosome organization in sperm is almost entirely non-random. The use of genomic clones allowed us to determine chromosome orientation and chromatin compaction in sperm. We found that in both species chromosomes maintain orientation of chromosomes in sperm independent of random or non-random positioning along the sperm nucleus. The distance of loci correlated with the total length of sperm nuclei, suggesting that chromatin extension depends on sperm elongation. In platypus, most sex chromosomes cluster in the posterior region of the sperm nucleus, presumably the result of postmeiotic association of sex chromosomes. Chicken and platypus autosomes sharing homology with the human X chromosome located centrally in both species suggesting that this is the ancestral position. This suggests that in some therian mammals a more anterior position of the X chromosome has evolved independently. PMID:18726609

  4. Dietary control of chromatin

    PubMed Central

    Huang, Zhiguang; Cai, Ling; Tu, Benjamin P

    2015-01-01

    Organisms must be able to rapidly alter gene expression in response to changes in their nutrient environment. This review summarizes evidence that epigenetic modifications of chromatin depend on particular metabolites of intermediary metabolism, enabling the facile regulation of gene expression in tune with metabolic state. Nutritional or dietary control of chromatin is an often-overlooked, yet fundamental regulatory mechanism directly linked to human physiology. Nutrient-sensitive epigenetic marks are dynamic, suggesting rapid turnover, and may have functions beyond the regulation of gene transcription, including pH regulation and as carbon sources in cancer cells. PMID:26094239

  5. Flow cytometry of sperm

    SciTech Connect

    Gledhill, B.L.

    1987-09-21

    This brief paper summarizes automated flow cytometric determination of sperm morphology and flow cytometry/sorting of sperm with application to sex preselection. In the latter context, mention is made of results of karyotypic determination of sex chromosome ratios in albumin-processed human sperm. 23 refs., 1 fig., 1 tab.

  6. Major regulatory mechanisms involved in sperm motility.

    PubMed

    Pereira, Rute; Sá, Rosália; Barros, Alberto; Sousa, Mário

    2017-01-01

    The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies.

  7. Major regulatory mechanisms involved in sperm motility

    PubMed Central

    Pereira, Rute; Sá, Rosália; Barros, Alberto; Sousa, Mário

    2017-01-01

    The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies. PMID:26680031

  8. The effects of pyridaben pesticide on the DNA integrity of sperms and early in vitro embryonic development in mice

    PubMed Central

    Ebadi Manas, Ghodrat; Hasanzadeh, Shapour; Najafi, Golamreza; Parivar, Kazem; Yaghmaei, Parichehr

    2013-01-01

    Background: Pyridaben, a pyridazinone derivative, is a new acaricide and insecticide for control of mites and some insects such as white flies, aphids and thrips. Objective: This study was designed to elucidate how pyridaben can affect the sperms' morphological parameters, its DNA integrity, and to estimate the effect of various quantities of pyridaben on in vitro fertilization rate. Materials and Methods: In this study, 80 adult male Balb/C strain mice were used. Animals were divided into control and two test groups. Control group received distilled water. The test group was divided into two subgroups, viz, high dose (212 mg/kg/day) and low dose (53 mg/kg/day) and they received the pyridaben, orally for duration of 45 days. The spermatozoa were obtained from caudae epididymides on day 45 in all groups. Sperm viability, protamin compression (nuclear maturity), DNA double-strand breaks, and in vitro fertilizing (IVF) ability were examined. Results: The pyridaben treatment provoked a significant decrease in sperm population and viability in epididymides. The data obtained from this experiment revealed that, the pyridaben brings about negative impact on the sperm maturation and DNA integrity in a time-dependent manner, which consequently caused a significant (p<0.05) reduction in IVF capability. Embryo developing arrest was significantly (p<0.05) higher in treated than the control group. Conclusion: Theses results confirmed that, the pyridaben is able to induce DNA damage and chromatin abnormalities in spermatozoa which were evident by low IVF rate. This article extracted from Ph.D. thesis. (Ghodrat Ebadi Mans) PMID:24639796

  9. Elemental composition of human semen is associated with motility and genomic sperm defects among older men

    PubMed Central

    Schmid, Thomas E.; Grant, Patrick G.; Marchetti, Francesco; Weldon, Rosana H.; Eskenazi, Brenda; Wyrobek, Andrew J.

    2013-01-01

    BACKGROUND Older men tend to have poorer semen quality and are generally at higher risks for infertility and abnormal reproductive outcomes. METHODS We employed proton-induced X-ray emission (PIXE, 3 MeV proton beam) to investigate the concentrations of zinc, copper, calcium, sulfur, chlorine, potassium, titanium, iron and nickel in washed sperm and seminal plasma from non-smoking groups of 10 older men (65–80 years old) and 10 younger men (22–28 years old) who were concurrently assayed for sperm function and genomicly defective sperm. RESULTS The older group showed elevated zinc, copper and calcium in sperm and elevated sulfur in seminal plasma compared with the younger men. The older group also showed reduced motility as well as increased sperm DNA fragmentation, achondroplasia mutations, DNA strand breaks and chromosomal aberrations. Sperm calcium and copper were positively associated with sperm DNA fragmentation (P < 0.03). Seminal sulfur was positively associated with sperm DNA fragmentation and chromosomal aberrations (P < 0.04), and negatively associated with sperm motility (P < 0.05). Sperm calcium was negatively associated with sperm motility, independent of male age (P = 0.01). CONCLUSIONS We identified major differences in elemental concentrations between sperm and seminal plasma and that higher sperm copper, sulfur and calcium are quantitatively associated with poorer semen quality and increased frequencies of genomic sperm defects. PMID:23042799

  10. Chromatin and DNA replication.

    PubMed

    MacAlpine, David M; Almouzni, Geneviève

    2013-08-01

    The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program.

  11. Archaeal chromatin proteins.

    PubMed

    Zhang, ZhenFeng; Guo, Li; Huang, Li

    2012-05-01

    Archaea, along with Bacteria and Eukarya, are the three domains of life. In all living cells, chromatin proteins serve a crucial role in maintaining the integrity of the structure and function of the genome. An array of small, abundant and basic DNA-binding proteins, considered candidates for chromatin proteins, has been isolated from the Euryarchaeota and the Crenarchaeota, the two major phyla in Archaea. While most euryarchaea encode proteins resembling eukaryotic histones, crenarchaea appear to synthesize a number of unique DNA-binding proteins likely involved in chromosomal organization. Several of these proteins (e.g., archaeal histones, Sac10b homologs, Sul7d, Cren7, CC1, etc.) have been extensively studied. However, whether they are chromatin proteins and how they function in vivo remain to be fully understood. Future investigation of archaeal chromatin proteins will lead to a better understanding of chromosomal organization and gene expression in Archaea and provide valuable information on the evolution of DNA packaging in cellular life.

  12. Analysis of Chromatin Organisation

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    Terms to be familiar with before you start to solve the test: chromatin, nucleases, sucrose density gradient centrifugation, melting point, gel electrophoresis, ethidium bromide, autoradiography, Southern blotting, Northern blotting, Sanger sequencing, restriction endonucleases, exonucleases, linker DNA, chloroform extraction, nucleosomes,…

  13. Morphofunctional disturbances of human sperm after incubation with organophosphorate pesticides.

    PubMed

    Contreras, H R; Badilla, J; Bustos-Obregón, E

    1999-08-01

    The organophosphorate pesticides are highly toxic for insects and mammals, but their effects in the male reproductive tract are scarcely known. Many alterations induced by organophosphorate pesticides have been described, such as: cytogenetic alterations in germinal cells, oligozoospermia and teratozoospermia in the mouse. Parathion, the pesticide mostly utilized in Chilean agriculture, is rapidly metabolized to paraoxon, the active metabolite, in mammalian organisms. The purpose of this study is to evaluate the effect of Parathion and paraoxon on different morphological and functional parameters of the sperm. Human spermatozoa were incubated with Parathion and paraoxon at different concentrations (0.05, 0.1, 0.2, 0.4 and 0.8 mM). Vitality (tripan blue and eosin tests), acrosome reaction (triple stain test), plasma membrane integrity (HOS-test), and chromatin stability (sodium thioglycolate test) were determined. The observations were done by optical microscopy at 1000x of magnification and three hundred sperms were evaluated for each treatment. The results indicated that Parathion and paraoxon increase the percent of sperm with acrosome reaction and also increase the percentage of sperm with chromatin decondensation in a dose-dependent manner. The vitality and plasma membrane integrity decrease significantly in a dose-dependent manner. The results suggest a direct action of Parathion and paraoxon on the different parameters studied. The morphofunctionality of sperm is altered significatively, suggesting that Parathion and paraoxon, thanks to their alkylating and electrophylic properties, could act on DNA and proteins respectively, to elicit these changes.

  14. Arabidopsis seed germination responses to osmotic stress involve the chromatin modifier PICKLE.

    PubMed

    Belin, Christophe; Lopez-Molina, Luis

    2008-07-01

    pkl mutant seed germination is hypersensitive to ABA treatment due to abnormally high and persistent ABI3 and ABI5 expression. PKL, a putative chromatin modifier, is instrumental to associate ABI3 and ABI5 with silent chromatin in response to ABA. Thus, PKL prevents exaggerated germination arrest responses by shutting off ABI3 and ABI5 expression in response to mild stresses.

  15. [Studies on the ploymorphic of sperm of F2 hybrids of red crucian carp x common carp].

    PubMed

    Li, Jian Zhong; Liu, Shao Jun; Zhang, Xuan Jie; Feng, Hao; Liu, Yun

    2004-08-01

    AThe ultrastructures of the sperm of F2 hybrids of red crucian carp x common carp were studied by using scanning and transmission electron microscope. The sperm of the F2 hybrids consisted of head, mid-piece and tail. There was no acrosome at the anterior end of the nuclears, whereas there was a vesicle. The results revealed that there existed obviously ploymorphic in the sperm of F2 hybrids. In the water-like semen from males of F2 hybrids, different sizes of the head of the sperm including haploid, diploid, tetraploid, and aneuploid sperm were observed. The head diameter of the smallest sperm was only 1.32 microm, but that of the biggest one was about 18.39 microm, and most of them varied from 1.85 to 2.15 microm. The haploid sperm was normal, while the a-neuploid, diploid, tetraploid and multiploid sperm were abnormal. Among the abnormal sperm, there was a super sperm with about 20 tails, whose head volume was much bigger than that of any other sperm. From the results of the transmission electron microscope, 3 sperm with two nucleus and 1 sperm with two tails were found. This study provided an useful evidence for the mechanism that the formation of tetraploid in F3 hybrids was due to the fertilization of the diploid eggs and diploid sperm produced by F2 hybrids.

  16. The use of complimentary assays to evaluate the enrichment of human sperm quality in asthenoteratozoospermic and teratozoospermic samples processed with Annexin-V magnetic activated cell sorting.

    PubMed

    Delbes, G; Herrero, M B; Troeung, E-T; Chan, P T K

    2013-09-01

    Sperm chromatin integrity may affect the outcomes of assisted reproductive technology (ART). Developing a clinically reliable strategy to enrich sperm samples with high chromatin quality spermatozoa prior to sperm banking or use in ART would thus be advantageous. The objectives of this study were to: (i) assess the sperm chromatin quality in men with different categories of semen parameters; and (ii) evaluate the extents of Annexin-V magnetic-activated cell sorting (MACS) technology coupled with differential density gradient centrifugation (DGC) in improving sperm chromatin quality. Three categories of men from couples attending a university-based fertility clinic were recruited based on their semen parameters: normozoospermic (n = 13), asthenoteratozoospermic (n = 17) and teratozoospermic (n = 12). For each patient, spermatozoa in semen samples were processed first by DGC to enrich the motility and further by MACS to remove spermatozoa showing apoptotic features. The yield and enrichment of sperm quality was evaluated at each step with conventional semen parameters in conjunction with a combination of five complementary assays, to assess sperm maturity, chromatin structure, compaction and DNA integrity (Hyaluronic Binding Assay, SCSA, chromomycine A3 staining and TUNEL and COMET assays). Our results demonstrated that, compared with normozoospermic samples, raw asthenoteratozoospermic and teratozoospermic samples had a higher proportion of spermatozoa containing DNA breaks, but only asthenoteratozoospermic exhibited altered chromatin structure and decreased binding to hyaluronic acid. Interestingly, the DGC appeared to select for more mature spermatozoa with high DNA compaction. More importantly, in all categories of semen samples, Annexin-V MACS allows enrichment of spermatozoa with good chromatin quality as measured by the TUNEL and SCSA. Because effective treatment modalities to improve sperm DNA damage are limited, our results suggest a potential clinical

  17. Acetylproteomic analysis reveals functional implications of lysine acetylation in human spermatozoa (sperm).

    PubMed

    Yu, Heguo; Diao, Hua; Wang, Chunmei; Lin, Yan; Yu, Fudong; Lu, Hui; Xu, Wei; Li, Zheng; Shi, Huijuan; Zhao, Shimin; Zhou, Yuchuan; Zhang, Yonglian

    2015-04-01

    Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed

  18. Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: examples from mollusks.

    PubMed

    Chiva, M; Saperas, N; Ribes, E

    2011-12-01

    In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and

  19. Sperm nuclei glutathione peroxidases and their occurrence in animal species with cysteine-containing protamines.

    PubMed

    Bertelsmann, Holger; Kuehbacher, Markus; Weseloh, Gundolf; Kyriakopoulos, Antonios; Behne, Dietrich

    2007-10-01

    The selenoenzyme sperm nuclei glutathione peroxidase (snGPx), also called the nuclear form of phospholipid hydroperoxide glutathione peroxidase (n-PHGPx), was found to be involved in the stabilization of condensed sperm chromatin, most likely by thiol to disulfide oxidation of the cysteine residues of the mammalian protamines, small nuclear basic proteins in the nuclei of sperm cells. By applying Acidic Urea-PAGE in combination with SDS-PAGE, snGPx with an apparent molecular mass of 34 kDa and a 24-kDa protein were purified from rat sperm nuclei. The 24-kDa protein was identified by means of mass spectrometry as a truncated form of snGPx produced by cleavage at the N-terminal end. After defined processing of spermatozoa and detergent treatment of the sperm nuclei fraction, snGPx and its truncated form were shown to be the only selenoproteins present in mature mammalian sperm nuclei. Both forms were found in mature rat and horse sperm nuclei but in man only snGPx was detected. In trout and chicken, species with sperm cells which likewise undergo chromatin condensation but do not contain cysteine in their protamines, the snGPx proteins were missing. This can be taken as an indirect proof of the function of snGPx to act as protamine cysteine thiol peroxidase in the mammalian species with cysteine-containing protamines.

  20. Association between sperm DNA integrity and seminal plasma antioxidant levels in health workers occupationally exposed to ionizing radiation

    SciTech Connect

    Kumar, Dayanidhi; Salian, Sujith Raj; Kalthur, Guruprasad; Uppangala, Shubhashree; Kumari, Sandhya; Challapalli, Srinivas; Chandraguthi, Shrinidhi Gururajarao; Jain, Navya; Krishnamurthy, Hanumanthappa; Kumar, Pratap; Adiga, Satish Kumar

    2014-07-15

    There is a paucity of data regarding the association between occupational radiation exposure and risk to human fertility. Recently, we provided the first evidence on altered sperm functional characteristics, DNA damage and hypermethylation in radiation health workers. However, there is no report elucidating the association between seminal plasma antioxidants and sperm chromatin integrity in occupationally exposed subjects. Here, we assessed the seminal plasma antioxidants and lipid peroxidation level in 83 men who were occupationally exposed to ionizing radiation and then correlated with the sperm chromatin integrity. Flow cytometry based sperm chromatin integrity assay revealed a significant decline in αt value in the exposed group in comparison to the non-exposed group (P<0.0001). Similarly, both total and reduced glutathione levels and total antioxidant capacity in the seminal plasma were significantly higher in exposed group than the non-exposed group (P<0.01, 0.001 and 0.0001, respectively). However, superoxide dismutase level and malondialdehyde level, which is an indicator of lipid peroxidation in the seminal plasma, did not differ significantly between two groups. The total antioxidant capacity (TAC) and GSH level exhibited a positive correlation with sperm DNA integrity in exposed subjects. To conclude, this study distinctly shows that altered sperm chromatin integrity in radiation health workers is associated with increase in seminal plasma antioxidant level. Further, the increased seminal plasma GSH and TAC could be an adaptive measure to tackle the oxidative stress to protect genetic and functional sperm deformities in radiation health workers. - Highlights: • Seminal plasma antioxidants were measured in men occupationally exposed to radiation. • Sperm chromatin integrity was significantly affected in the exposed group. • Glutathione and total antioxidant capacity was significantly higher in exposed group. • Sperm DNA damage in exposed subjects

  1. Meiotic chromosome abnormalities in human spermatogenesis.

    PubMed

    Martin, Renée H

    2006-08-01

    The last few years have witnessed an explosion in the information about chromosome abnormalities in human sperm and the meiotic events that predispose to these abnormalities. We have determined that all chromosomes are susceptible to nondisjunction, but chromosomes 21 and 22 and, especially, the sex chromosomes have an increased frequency of aneuploidy. Studies are just beginning on the effects of potential mutagens on the chromosomal constitution of human sperm. The effects of pesticides and cancer therapeutic agents have been reviewed. In the last decade, there has been a great impetus to study chromosome abnormalities in sperm from infertile men because the advent of intracytoplasmic sperm injection (ICSI) made it possible for these men to father pregnancies. A large number of studies have demonstrated that infertile men have an increased frequency of chromosomally abnormal sperm and children, even when they have a normal somatic karyotype. Meiotic studies on the pachytene stage of spermatogenesis have demonstrated that infertile men have impaired chromosome synapsis, a significantly decreased frequency of recombination, and an increased frequency of chromosomes completely lacking a recombination site. Such errors make these cells susceptible to meiotic arrest and the production of aneuploid gametes.

  2. Mammalian sperm morphometry.

    PubMed Central

    Gage, M J

    1998-01-01

    Understanding the adaptive significance of sperm form and function has been a challenge to biologists because sperm are highly specialized cells operating at a microscopic level in a complex environment. A fruitful course of investigation has been to use the comparative approach. This comparative study attempts to address some fundamental questions of the evolution of mammalian sperm morphometry. Data on sperm morphometry for 445 mammalian species were collated from published sources. I use contemporary phylogenetic analysis to control for the inherent non-independence of species and explore relationships between the morphometric dimensions of the three essential spermatozoal components: head, mid-piece and flagellum. Energy for flagellar action is metabolized by the mitochondrial-dense mid-piece and these combine to propel the sperm head, carrying the male haplotype, to the ovum. I therefore search for evolutionary associations between sperm morphometry and body mass, karyotype and the duration of oestrus. In contrast to previous findings, there is no inverse correlation between body weight and sperm length. Sperm mid-piece and flagellum lengths are positively associated with both head length and area, and the slopes of these relationships are discussed. Flagellum length is positively associated with mid-piece length but, in contrast to previous research and after phylogenetic control, I find no relationship between flagellum length and the volume of the mitochondrial sheath. Sperm head dimensions are not related to either genome mass or chromosome number, and there are no relationships between sperm morphometry and the duration of oestrus. PMID:9474794

  3. Relationship between the nuclear morphology of the sperm of 10 bulls and their fertility.

    PubMed

    Vieytes, A L; Cisale, H O; Ferrari, M R

    2008-11-22

    The relationships between the fertility and nuclear morphology, chromatin maturity and chromatin condensation of the sperm of three bulls with a calving rate over a year of more than 65 per cent, four bulls with a calving rate between 65 per cent and 35 per cent, and three bulls with a calving rate of less than 35 per cent were studied. The sperm nuclei were stained with the Feulgen reaction, and chromatin condensation and maturation were evaluated in situ by staining with toluidine blue and acid aniline blue. Nuclear chromatin decondensation was induced with dithiothreitol; this showed that in the bulls with low fertility, more than 35 per cent of nuclei were decondensed, and that one of them had the lowest percentage of normal nuclei (64.9 per cent) and stronger positive reactions to the acid aniline blue and toluidine blue stains than the other bulls.

  4. Quantification of mammalian sperm morphology by slit-scan flow cytometry

    SciTech Connect

    Benaron, D.A.; Gray, J.W.; Gledhill, B.L.; Lake, S.; Wyrobek, A.J.; Young, I.T.

    1982-03-01

    The head shapes of mammalian sperm have been measured by slit-scan flow cytometry (SSFCM). In this approach, the distribution of fluorescence along acriflavine stained mammalian sperm is recorded and used as a measure of head shape. Fluorescence profiles were measured for sperm from mice, rabbits, hamsters, and bulls, and for sperm from mice exposed to testicular x-irradiation from 0 to 900 rads. The profiles for sperm from nonirradiated animals were characteristic of each species and were reproducible from sperm to sperm. Some of the fluorescence profiles for sperm from the irradiated mice differed significantly from the profiles usually measured for sperm from exposed mice. An algorithm was developed to determine the frequency of these sperm. The estimated frequencies of atypical profiles correlated well (r . 0.99) with the frequencies of abnormally shaped sperm determined by microscopic scoring. The maximum SSFCM sensitivity (minimum detectable dose . 199 rad) was not as high as that for the visual assay (minimum detectable dose . 116 rad). However, only 100 profiles were measured by SSFCM at each dose while at least 500 sperm were scored visually at each dose. The sensitivity of the SSFCM assay should be increased substantially by measuring more profiles. The objective nature of SSFCM couple with the high correlation with results from the visually based assay of morphology suggests the use of SSFCM to measure frequencies of misshapen sperm when testing for mutagens or monitoring for effects of environmental contaminants.

  5. Oral antioxidant treatment partly improves integrity of human sperm DNA in infertile grade I varicocele patients.

    PubMed

    Gual-Frau, Josep; Abad, Carlos; Amengual, María J; Hannaoui, Naim; Checa, Miguel A; Ribas-Maynou, Jordi; Lozano, Iris; Nikolaou, Alexandros; Benet, Jordi; García-Peiró, Agustín; Prats, Juan

    2015-09-01

    Infertile males with varicocele have the highest percentage of sperm cells with damaged DNA, compared to other infertile groups. Antioxidant treatment is known to enhance the integrity of sperm DNA; however, there are no data on the effects in varicocele patients. We thus investigated the potential benefits of antioxidant treatment specifically in grade I varicocele males. Twenty infertile patients with grade I varicocele were given multivitamins (1500 mg L-Carnitine, 60 mg vitamin C, 20 mg coenzyme Q10, 10 mg vitamin E, 200 μg vitamin B9, 1 μg vitamin B12, 10 mg zinc, 50 μg selenium) daily for three months. Semen parameters including total sperm count, concentration, progressive motility, vitality, and morphology were determined before and after treatment. In addition, sperm DNA fragmentation and the amount of highly degraded sperm cells were analyzed by Sperm Chromatin Dispersion. After treatment, patients showed an average relative reduction of 22.1% in sperm DNA fragmentation (p = 0.02) and had 31.3% fewer highly degraded sperm cells (p = 0.07). Total numbers of sperm cells were increased (p = 0.04), but other semen parameters were unaffected. These data suggest that sperm DNA integrity in grade I varicocele patients may be improved by oral antioxidant treatment.

  6. Detection of aneuploid human sperm by fluorescence in situ hybridization: Evidence for a donor difference in frequency of sperm disomic for chromosomes 1 and Y

    SciTech Connect

    Robbins, W.A. Lawrence Livermore National Lab., CA ); Segraves, R.; Pinkel, D. ); Wyrobek, A.J. )

    1993-04-01

    Fluorescence in situ hybridization with repetitive-sequence DNA probes was used to detect human sperm disomic for chromosomes 1 and Y in three healthy men. Data on these same men had been obtained previously, using the human-sperm/hamster-egg cytogenetic technique, providing a cytogenetic reference for validating sperm hybridization measurements. Air-dried smears were prepared from semen samples and treated with DTT and lithium diiodosalicylate to expand sperm chromatin. Hybridization with fluorescently tagged DNA probes for chromosomes 1 (pUC177) or Y (pY3.4) yielded average frequencies of sperm with two fluorescent domains of 14.2[+-]2.4/10,000 and 5.6[+-]1.6/10,000 sperm, respectively. These frequencies did not differ statistically from frequencies of hyperploidy observed for these chromosomes with the hamster technique. In addition, frequencies of disomic sperm from one donor were elevated [approximately]2.5-fold above those of other donors, for both chromosomes 1 (P = .045) and Y (P = .01), consistent with a trend found with the hamster technique. The authors conclude that fluorescence in situ hybridization to sperm chromosomes provides a valid and promising measure of the frequency of disomic human sperm. 43 refs., 1 fig., 4 tabs.

  7. Prognosis for sperm fertilizability: analysis of different variables in men.

    PubMed

    Check, J H; Check, M L; Katsoff, D

    2002-01-01

    An overview of various sperm tests is presented. The standard semen analysis obtained by most clinicians evaluating infertility usually consists of sperm concentration, percent motility, quality of motility, and sperm morphology. Unfortunately, unless the motile density is extremely low, the count and motility are not good prognosticators of fertility potential. Values above the norm for normal fertile couples unfortunately cannot reliably predict normal fertility potential. It is important to find sperm tests that are easy to perform, are relatively inexpensive, and provide an accurate prognosis. Strict morphology was hoped to be such a tool with initial optimism that it was far superior to standard morphology. Unfortunately, this test also failed to be the ideal inexpensive prognostic test after further evaluation. One test that is inexpensive and highly correlates with fertilizability is the presence of antisperm antibodies since their presence frequently does not alter count, motility, or morphology. This test should be performed as part of the routine semen analysis. Other tests highly correlate with the achievement of pregnancy and are simple and inexpensive to perform, but, interestingly, do not correlate with fertilizability. These include the hypoosmotic swelling test (HOST) and the sperm stress test. Abnormalities in these tests imply a different abnormality of sperm that leads to conception failure and that is the transfer of a toxic factor from the sperm to oocyte to embryo that prevents the embryo from implanting. Certainly, the simple, inexpensive HOST should be performed routinely. Other tests of sperm function, e.g., sperm penetration assay, sperm zona pellucida binding assay, and acrosome reaction, have their definite place in the evaluation of the infertile male. However, because they are expensive and difficulty to perform they lend themselves to certain specific circumstances but not to routine testing.

  8. Role of prostatic fluid in cooled canine epididymal sperm.

    PubMed

    Hori, T; Masuda, T; Kobayashi, M; Kawakami, E

    2017-03-29

    In this study, sperms collected from the right and left cauda epididymis were grouped into having canine prostatic fluid (PF) sensitization or not diluted with egg yolk Tris-fructose citrate extender, and stored at 4°C. The necessity of canine PF in cooled preservation was determined by elucidating the sperm quality after the storage. As a result, while there was no difference among all groups up to 48 hr of storage, after storage for 96 hr and more, a significantly lower sperm motility was observed in the group without being sensitized to PF than the groups with being sensitized to PF (p < .05, p < .01). Although sperm abnormality increased in all groups with increased storage time, the group without being sensitized to PF showed significantly higher sperm abnormality than did the groups with being sensitized to PF after storage for 24 hr and more (p < .01). From these findings, we concluded that PF was necessary for the cooled preservation of the canine sperm because these sperms were protected from any effects of low temperatures by being sensitized to PF.

  9. Alveolar abnormalities

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/001093.htm Alveolar abnormalities To use the sharing features on this page, please enable JavaScript. Alveolar abnormalities are changes in the tiny air sacs in ...

  10. Nail abnormalities

    MedlinePlus

    Beau's lines; Fingernail abnormalities; Spoon nails; Onycholysis; Leukonychia; Koilonychia; Brittle nails ... 2012:chap 71. Zaiac MN, Walker A. Nail abnormalities associated with systemic pathologies. Clin Dermatol . 2013;31: ...

  11. Metabolic activity of sperm cells: correlation with sperm cell concentration, viability and motility in the rabbit.

    PubMed

    Sabés-Alsina, Maria; Planell, Núria; Gil, Sílvia; Tallo-Parra, Oriol; Maya-Soriano, Maria José; Taberner, Ester; Piles, Miriam; Sabés, Manel; Lopez-Bejar, Manel

    2016-10-01

    The resazurin reduction test (RRT) is a useful technique to assess the metabolic rate of sperm cells. RRT depends on the ability of metabolically active cells to reduce the non-fluorescent dye resazurin to the fluorescent resorufin. The aim of this study was to develop a vital fluorometric method to evaluate metabolic activity of rabbit sperm cells. Twenty-five rabbit males were included in the study. Viability and morphology, motility and metabolic activity were evaluated using an eosin-nigrosin staining, a computer-assisted semen analysis (CASA) and the RRT, respectively. Spearman rank correlation analysis was used to determine the correlation between RRT and semen parameters. After evaluation, a concentration of 10 × 106 sperm cells/ml was selected for further experiments with RRT. No significant correlation was found between the RRT results and the motility parameters. However, after RRT a significant positive correlation between relative fluorescence units and the percentage of alive spermatozoa (r = 0.62; P = 0.001) and a negative one with the percentage of sperm cells with acrosomic abnormalities (r = -0.45; P < 0.05) were detected. The vital assessment of metabolic rate of sperm cells by RRT could provide more information about semen quality than other routine semen analysis, correlating with sperm viability and acrosome status information.

  12. Morphological study of boar sperm during their passage through the female genital tract

    PubMed Central

    GARCÍA-VÁZQUEZ, Francisco Alberto; HERNÁNDEZ-CARAVACA, Iván; MATÁS, Carmen; SORIANO-ÚBEDA, Cristina; ABRIL-SÁNCHEZ, Silvia; IZQUIERDO-RICO, María José

    2015-01-01

    Once deposited in the female tract, sperm face a series of challenges that must be overcome to ensure the presence of an adequate normal sperm population close to the site of fertilization. Our aim was to evaluate the influence of the uterine milieu on boar sperm morphology. In experiment 1, sperm morphology was evaluated in the backflow (60 min after insemination) and within the uterotubal junction (UTJ) (collected ~24 h after insemination) following intrauterine sperm deposition (n = 6) and compared with the morphology of the sperm in the insemination dose. In experiment 2, the influence of the uterine fluid (UF) on sperm morphological modifications was evaluated. For this purpose, ejaculated (n = 4) and epididymal (n = 4) sperm were in vitro incubated with or without UF for 2 and 24 h. In both experiments, sperm were classified as normal, having a cytoplasmic droplet (proximal or distal) or having tail defects. The results of experiment 1 pointed to an increase in morphologically abnormal sperm collected in the backflow (27.70%) and a reduction of the same in the UTJ (2.12%) compared with the insemination dose (17.75%) (P < 0.05). In experiment 2, incubation of ejaculated sperm with UF did not provoke any morphological modifications; however, when epididymal sperm were incubated with UF, a pronounced increase in the percentage of normal sperm was evident after 24 h compared with the initial dose (from 25.77% to 53.58%, P < 0.05), mainly due to distal cytoplasmatic droplet shedding (53.22 vs. 20.20%). In conclusion, almost all the sperm that colonize the UTJ had a normal morphology, with part of the abnormal sperm having been discarded in the backflow and part selected/modified on their way to the oviduct. UF seems to influence cytoplasmic distal droplet removal, as demonstrated previously in seminal plasma. PMID:26119829

  13. EVALUATION OF SPERM CHROMATIN STRUCTURE ASSAY (SCSA REGISTERED TRADEMARK) IN HUMAN SPERM AFTER SIMULATED OVERNIGHT SHIPMENT

    EPA Science Inventory

    Home semen collection kits allow men to collect a sample at their convenience and send it via overnight mail to the laboratory. Benefits of this approach include facilitated sample collection from different geographic locations, minimized variability through analysis by a central...

  14. Cas9 Functionally Opens Chromatin

    PubMed Central

    Barkal, Amira A.; Srinivasan, Sharanya; Hashimoto, Tatsunori; Gifford, David K.; Sherwood, Richard I.

    2016-01-01

    Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding. PMID:27031353

  15. Molecular Toxicology of Chromatin

    DTIC Science & Technology

    1992-01-01

    FINAL 01 Jan 89 TO 31 Dec 91 4. ITL ANO SUS Y, L RE %UMAS MOLECULAR TOXICOLOGY OF CHROMATIN AFOSR-89-0231 PE - 61102F AUT PR - 2312 TA - A5 Dr Ernest Kun...Waterbury, CT), 2-mercaptoethanol, NAD+, NADPH, nucleo- tides, sodium tungstate , hydrogen peroxide, Tris and MES buffers from Sigma (St. Louis, MO...ml) with sodium tungstate (5.93 g, in 20 ml H20) for 1.5 h followed by extraction of the green product into ethyl acetate, washing with 0.1 N HCl, and

  16. Trade-off between carotenoid-based sexual ornamentation and sperm resistance to oxidative challenge.

    PubMed

    Tomášek, Oldřich; Albrechtová, Jana; Němcová, Martina; Opatová, Pavlína; Albrecht, Tomáš

    2017-01-25

    It has been hypothesized that carotenoid-based sexual ornamentation signals male fertility and sperm competitive ability as both ornamentation and sperm traits may be co-affected by oxidative stress, resulting in positive covariation (the 'redox-based phenotype-linked fertility hypothesis'; redox-based PLFH). On the other hand, the 'sperm competition theory' (SCT) predicts a trade-off between precopulatory and postcopulatory traits. Here, we manipulate oxidative status (using diquat dibromide) and carotenoid availability in adult zebra finch (Taeniopygia guttata) males in order to test whether carotenoid-based beak ornamentation signals, or is traded off against, sperm resistance to oxidative challenge. Initial beak colouration, but not its change during the experiment, was associated with effect of oxidative challenge on sperm velocity, such that more intense colouration predicted an increase in sperm velocity under control conditions but a decline under oxidative challenge. This suggests a long-term trade-off between ornament expression and sperm resistance to oxidative challenge. Shortening of the sperm midpiece following oxidative challenge further suggests that redox homeostasis may constrain sperm morphometry. Carotenoid supplementation resulted in fewer sperm abnormalities but had no effect on other sperm traits. Overall, our data challenge the redox-based PLFH, partially support the SCT and highlight the importance of carotenoids for normal sperm morphology.

  17. Changes of sperm quality and hormone receptors in the rat testis after exposure to methamphetamine.

    PubMed

    Nudmamud-Thanoi, Sutisa; Sueudom, Wanvipa; Tangsrisakda, Nareelak; Thanoi, Samur

    2016-10-01

    Methamphetamine (METH) is known to damage neurons and induce psychosis. It can also induce apoptosis in seminiferous tubules and affect sperm quality. The present study was carried out to investigate the effect of a rat model of METH addiction on sperm quality and expression of progesterone receptors (PR) and estrogen receptors (ER) in the testis. Sperm quality parameters including sperm motility, sperm morphology and sperm concentration were examined. Protein and gene expressions PR, ERα and ERβ were studied using immunohistochemistry and reverse transcriptase-polymerase chain reaction, respectively. The percentages of normal sperm motility and normal sperm morphology were significantly decreased in animals receiving METH, especially in escalating dose (ED METH) and escalating dose-binge (ED-binge METH) groups when compared with control. In addition, sperm concentrations in ED METH and ED-binge METH groups were numerically decreased. PR, ERα and ERβ immunoreactive cells were significantly decreased in spermatogonia, spermatogenic cells and especially in Sertoli cells in all METH-treated groups. Furthermore, messenger RNA expression of PR, ERα and ERβ were also significantly decreased in all METH-treated animals. These results indicate that METH can induce abnormal sperm quality. These changes of sperm quality may relate to the reduction of PR, ERα and ERβ expressions in male germ cells and Sertoli cells which are essential for spermatogenesis and development of sperm.

  18. Dynamics of Male and Female Chromatin during Karyogamy in Rice Zygotes1[C][W][OPEN

    PubMed Central

    Ohnishi, Yukinosuke; Hoshino, Rina; Okamoto, Takashi

    2014-01-01

    In angiosperms, the conversion of an egg cell into a zygote involves two sequential gametic processes: plasmogamy, the fusion of the plasma membranes of male and female gametes, and karyogamy, the fusion of the gametic nuclei. In this study, the nuclei and nuclear membranes of rice (Oryza sativa) gametes were fluorescently labeled using histones 2B-green fluorescent protein/red fluorescent protein and Sad1/UNC-84-domain protein2-green fluorescent protein, respectively, which were heterologously expressed. These gametes were fused in vitro to produce zygotes, and the nuclei and nuclear membranes in the zygotes were observed during karyogamy. The results indicated that the sperm nucleus migrates adjacent to the egg nucleus 5 to 10 min after plasmogamy via an actin cytoskelton, and the egg chromatin then appears to move unidirectionally into the sperm nucleus through a possible nuclear connection. The enlargement of the sperm nucleus accompanies this possible chromatin remodeling. Then, 30 to 70 min after fusion, the sperm chromatin begins to decondense with the completion of karyogamy. Based on these observations, the development of early rice zygotes from plasmogamy to karyogamy was divided into eight stages, and using reverse transcription PCR analyses, paternal and de novo synthesized transcripts were separately detected in zygotes at early and late karyogamy stages, respectively. PMID:24948834

  19. Quantitative methods of measuring the sensitivity of the mouse sperm morphology assay

    SciTech Connect

    Moore, D.H.; Bennett, D.E.; Kranzler, D.; Wyrobek, A.J.

    1982-09-01

    In this study murine sperm were subjected to graded doses of X irradiation (0 to 120 rad) to determine whether quantitative measurements made on enlarged photographs of the sperm heads are related to radiation dose. We found that the Mahalanobis distance statistic, when used to measure distance in a multivariate space from a control group of measurements, could be used to classify sperm as normal or abnormal. The percent classified as abnormal by this method was found to be linearly related to dose. The results suggest that sensitivity of the murine sperm assay can be improved by selecting an optimal set of measurements. This improvement can reduce the doubling dose from approximately 70 rad to 10 to 15 rad while keeping the percentage of abnormal sperm in control mice at 3%, equal to the current visual method.

  20. Impact of kudzu and puerarin on sperm function.

    PubMed

    Gray, Sandra L; Lackey, Brett R; Boone, William R

    2015-06-01

    The goal of this study was to investigate the impact of kudzu (Pueraria mirifica) and the isoflavone puerarin in functional toxicological tests on spermatozoa and to assess the affinity of extracts and pure isoflavones for estrogen receptor (ER)-alpha and -beta (ERα, ERβ) in receptor binding assays. Capacitation, acrosome reaction and chromatin decondensation in spermatozoa were analyzed using microscopic analysis. Kudzu, but not puerarin, reduced motility of sperm. Puerarin reduced the percent spontaneous acrosome reaction in spermatozoa. The pathways used by kudzu that affect sperm function are not fully mirrored by puerarin. Puerarin, kudzu and its other phytoestrogenic components displayed preferential affinity for ERβ, however the diverse effects of kudzu and puerarin on sperm function implicate the involvement of multiple signaling systems.

  1. Mapping chromatin modifications in nanochannels

    NASA Astrophysics Data System (ADS)

    Lim, Shuang Fang; Karpusenko, Alena; Riehn, Robert

    2013-03-01

    DNA and chromatin are elongated to a fixed fraction of their contour length when introduced into quasi-1d nanochannels. Because single molecules are analyzed, their hold great potential for the analysis for the genetic analysis of material from single cells. In this study, we have reconstituted chromatin with histones from a variety of sources, and mapped the modification profile of the chromatin. We monitored methylation and acetylation patterns of the histone tail protein residues using fluorescently labelled antibodies. Using those, we distinguished chromatin reconstituted from chicken erythrocytes, calf thymus, and HeLa cells. We discuss prospects for profiling histone modifications for whole chromosomes from single cells.

  2. Chromatin structure in barley nuclei.

    PubMed

    Mithieux, G; Roux, B

    1983-10-03

    In order to study the chromatin structure of a higher plant we used a high-yield method, which allows one to obtain up to 10(9) nuclei/kg fresh barley leaves. Significant amounts of low-ionic-strength-soluble chromatin can be extracted from these nuclei. Physicochemical properties were examined and discussed. Electric birefringence allowed us to observe the same transition in electro-optical properties as has been observed for animal chromatin, and suggested the existence of a symetrical structure occurring for approximately six nucleosomes. Circular dichroism showed that barley oligonucleosomes exhibit a higher molar ellipticity at 282 nm than total soluble chromatin and than their animal counterparts.

  3. Sperm Patch-Clamp

    PubMed Central

    Lishko, Polina; Clapham, David E.; Navarro, Betsy; Kirichok, Yuriy

    2014-01-01

    Sperm intracellular pH and calcium concentration ([Ca2+]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca2+] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels. PMID:23522465

  4. Increased chromatin fragmentation and reduced acrosome integrity in spermatozoa of red deer from lead polluted sites.

    PubMed

    Castellanos, Pilar; del Olmo, Enrique; Fernández-Santos, M Rocío; Rodríguez-Estival, Jaime; Garde, J Julián; Mateo, Rafael

    2015-02-01

    Vertebrates are constantly exposed to a diffuse pollution of heavy metals existing in the environment, but in some cases, the proximity to emission sources like mining activity increases the risk of developing adverse effects of these pollutants. Here we have studied lead (Pb) levels in spermatozoa and testis, and chromatin damage and levels of endogenous antioxidant activity in spermatozoa of red deer (Cervus elaphus) from a Pb mining area (n=37) and a control area (n=26). Deer from the Pb-polluted area showed higher Pb levels in testis parenchyma, epididymal cauda and spermatozoa, lower values of acrosome integrity, higher activity of glutathione peroxidase (GPx) and higher values of DNA fragmentation (X-DFI) and stainability (HDS) in sperm than in the control area. These results indicate that mining pollution can produce damage on chromatin and membrane spermatozoa in wildlife. The study of chromatin fragmentation has not been studied before in spermatozoa of wildlife species, and the sperm chromatin structure assay (SCSA) has been revealed as a successful tool for this purpose in species in which the amount of sperm that can be collected is very limited.

  5. New method for sperm evaluation by 3-dimensional laser scanning microscopy in different laboratory animal species.

    PubMed

    Weber, Klaus; Waletzky, Alexander; Fendl, Diana; Ordóñez, Patricia; Takawale, Pradeep; Hein, Felix; Riedel, Wolfram; König, Andres; Kunze, Marc; Leoni, Anne-Laure; Rivera, Javier; Quirici, Roberto; Romano, Ivano; Paepke, Susanne; Okazaki, Yoshimasa; Hardisty, Jerry F

    2014-01-01

    Sperm analysis is one of the end points in reproductive toxicology studies. Different methods for quantitative sperm analysis have been described. For qualitative morphological sperm analysis, either such techniques or smears of sperm and histological sperm staging are in use. Any of these methods provides morphological results on a light microscopy level. Laser scanning microscopy is a technique using a focused laser for scanning an object. The Olympus 3D Laser Scanning Microscope LEXT OLS4000 with optional possibilities of differential interference contrast provides a microscopic method for visualizing microasperities, which are far beyond the resolving power of a typical light or laser microscope. This technique was applied to sperm of mice, rats, rabbits, and cynomolgus monkeys at magnifications up to ×17 090. The obtained images are comparable to those of a scanning electron microscope under relatively low-power magnifications. Measurements on sperm parameters were taken by an integrated image analysis software tool. Abnormalities were easily detectable.

  6. Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

    PubMed Central

    Cortés-Gutiérrez, Elva I.; López-Fernández, Carmen; Fernández, José Luis; Dávila-Rodríguez, Martha I.; Johnston, Stephen D.; Gosálvez, Jaime

    2014-01-01

    Key Concepts The two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell.Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage.Alkaline treatment may break the sugar–phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites “ALSs.”DBD–FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions.The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome.The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to

  7. Sperm structure and phylogeny of Astigmata.

    PubMed

    Liana, Marcin; Witaliński, Wojciech

    2005-09-01

    The Astigmata, a large and variable group, is still a subject of taxonomic dispute. Particularly, their origin from ancestors of the lower oribatid mites (e.g., Malaconothroidea) seems well documented by many lines of evidence. The structure of spermatozoa has been successfully applied to phylogenetic investigations in many animal groups. The aim of our study was to provide new data on spermatozoon structure in Astigmata and to consider its appropriateness in phylogenetic studies. The study reveals information on spermatozoa in 17 species of Astigmata (11 species studied for the first time) extending our knowledge to 18 species (one species known only from the literature) representing 12 families and 7 superfamilies. Spermatozoa have the same basic structure in all species: cells are multiform and the chromatin forms thin threads embedded directly in the cytoplasm; the acrosome is absent. The cytoplasm in most species contains electron-dense lamellae, varying in both number and arrangement within the cell. In Sarcoptoidea, electron-dense tubules in contact with lamellae margins were also observed in Psoroptidae (Psoroptes equi), whereas in two representatives of Sarcoptidae (Notoedres cati and Sarcoptes scabiei), only electron-dense tubules were found. In two species, Canestrinia sellnicki (Canestrinioidea: Canestriniidae) and Scutulanyssus obscurus (Analgoidea: Pteronyssidae), neither lamellae nor tubules were present. The mitochondria in a spermatozoon are usually gathered at the cell periphery and their structure is usually modified to form so-called mitochondrial derivatives. The chromatin threads are an autapomorphy strongly supporting the monophyly of Astigmata. As spermatozoa vary considerably between species in Astigmata, we deduce that sperm structure may be useful for phylogenetic analyses within the group. Several conclusions concerning the affinities within Astigmata are presented. Spermatology seems to be unhelpful, however, in questions on the origin

  8. Sperm ultrastructure of the spider crab Maja brachydactyla (Decapoda: Brachyura).

    PubMed

    Simeó, Carles G; Kurtz, Kathryn; Rotllant, Guiomar; Chiva, Manel; Ribes, Enric

    2010-04-01

    This study describes the morphology of the sperm cell of Maja brachydactyla, with emphasis on localizing actin and tubulin. The spermatozoon of M. brachydactyla is similar in appearance and organization to other brachyuran spermatozoa. The spermatozoon is a globular cell composed of a central acrosome, which is surrounded by a thin layer of cytoplasm and a cup-shaped nucleus with four radiating lateral arms. The acrosome is a subspheroidal vesicle composed of three concentric zones surrounded by a capsule. The acrosome is apically covered by an operculum. The perforatorium penetrates the center of the acrosome and has granular material partially composed of actin. The cytoplasm contains one centriole in the subacrosomal region. A cytoplasmic ring encircles the acrosome in the subapical region of the cell and contains the structures-organelles complex (SO-complex), which is composed of a membrane system, mitochondria with few cristae, and microtubules. In the nucleus, slightly condensed chromatin extends along the lateral arms, in which no microtubules have been observed. Chromatin fibers aggregate in certain areas and are often associated with the SO-complex. During the acrosomal reaction, the acrosome could provide support for the penetration of the sperm nucleus, the SO-complex could serve as an anchor point for chromatin, and the lateral arms could play an important role triggering the acrosomal reaction, while slightly decondensed chromatin may be necessary for the deformation of the nucleus.

  9. Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia.

    PubMed

    Vendrell, J M; García, F; Veiga, A; Calderón, G; Egozcue, S; Egozcue, J; Barri, P N

    1999-02-01

    The incidence of meiotic abnormalities and their relationship with different spermatogenic parameters was assessed in 103 male patients with presumably idiopathic severe oligoasthenozoospermia (motile sperm concentration < or = 1.5 x 10(6)/ml). Meiosis on testicular biopsies was independently evaluated by two observers. Meiotic patterns included normal meiosis and two meiotic abnormalities, i.e. severe arrest and synaptic anomalies. A normal pattern was found in 64 (62.1%), severe arrest in 21 (20.4%) and synaptic anomalies in 18 (17.5%). The overall rate of meiotic abnormalities was 37.9%. Most (66.7%) meiotic abnormalities occurred in patients with a sperm concentration < or = 1 x 10(6)/ml. In this group, total meiotic abnormalities were found in 57.8% of the patients; of these, 26.7% had synaptic anomalies. When the sperm concentration was < or = 0.5 x 10(6)/ml, synaptic anomalies were detected in 40% of the patients. In patients with increased follicle stimulating hormone (FSH) concentrations, total meiotic abnormalities occurred in 54.8% (synaptic anomalies in 22.6%). There were statistically significant differences among the three meiotic patterns in relation to sperm concentration (P < 0.001) and serum FSH concentration (P < 0.05). In the multivariate analysis, sperm concentration < or = 1 x 10(6)/ml and/or FSH concentration > 10 IU/l were the only predictors of meiotic abnormalities.

  10. Post-Translational Modifications of Histones in Human Sperm.

    PubMed

    Krejčí, Jana; Stixová, Lenka; Pagáčová, Eva; Legartová, Soňa; Kozubek, Stanislav; Lochmanová, Gabriela; Zdráhal, Zbyněk; Sehnalová, Petra; Dabravolski, Siarhei; Hejátko, Jan; Bártová, Eva

    2015-10-01

    We examined the levels and distribution of post-translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM-FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM-FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non-integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC-MS/MS and MALDI-TOF MS analysis. Taken together, these data indicate that particular post-translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual.

  11. Dynamics of sperm DNA fragmentation in the swine: ejaculate and temperature effects.

    PubMed

    Pérez-Llano, B; López-Fernández, C; García-Casado, P; Arroyo, F; Gosalbez, A; Sala, R; Gosálvez, J

    2010-06-01

    The dynamics of sperm DNA fragmentation was examined in 16 boar ejaculates using the sperm chromatin dispersion (SCD) test and a two-tail comet assay. The net sperm rich fraction was preserved at two different temperatures (Trial 1: 15 degrees C, n=10; Trial 2: 37 degrees C, n=6) and sub-samples were taken every day until a sperm motility of zero. Significant differences in the dynamics of DNA fragmentation were observed among the different ejaculates and also according to the storage temperature. After analyzing the dynamic response of the sperm DNA damage, when the sperm samples are incubated at 15 or 37 degrees C, each ejaculate could be classified and a considerable variation among individuals for an increase in DNA damage was observed. Thus, while in some ejaculates no rise in DNA fragmentation was observed, in others, the sperm DNA fragmentation process was triggered during the initial days of the experiment. In general, sperm incubation at 37 degrees C diminished sperm DNA quality. The two-tail comet assay indicated that at time zero existing DNA damage mainly consisted of double stranded DNA breakage. During storage, DNA damage affected one of the DNA strands until a second wave of DNA damage, in which there was both single and double stranded DNA damage.

  12. The nature of human sperm head vacuoles: a systematic literature review.

    PubMed

    Boitrelle, Florence; Guthauser, Bruno; Alter, Laura; Bailly, Marc; Wainer, Robert; Vialard, François; Albert, Martine; Selva, Jacqueline

    2013-01-01

    Motile sperm organelle morphology examination (MSOME) involves the use of differential interference contrast microscopy (also called Nomarski contrast) at high magnification (at least 6300x) to improve the observation of live human spermatozoa. In fact, this technique evidences sperm head vacuoles that are not necessarily seen at lower magnifications - particularly if the vacuoles are small (i.e. occupying <4% of the sperm head's area). However, a decade after MSOME's introduction, it is still not clear whether sperm head vacuoles are nuclear, acrosomal and/or membrane-related in nature. In an attempt to clarify this debate, we performed a systematic literature review in accordance with the PRISMA guidelines. The PubMed database was searched from 2001 onwards with the terms "MSOME", "human sperm vacuoles", "high-magnification, sperm". Out of 180 search results, 21 relevant English-language publications on the nature of human sperm head vacuoles were finally selected and reviewed. Our review of the literature prompted us to conclude that sperm-head vacuoles are nuclear in nature and are related to chromatin condensation failure and (in some cases) sperm DNA damage.

  13. Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

    PubMed Central

    Kuhtz, Juliane; Schneider, Eberhard; El Hajj, Nady; Zimmermann, Lena; Fust, Olga; Linek, Bartosz; Seufert, Rudolf; Hahn, Thomas; Schorsch, Martin; Haaf, Thomas

    2014-01-01

    The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1–14% GTL2 epimutations. PMID:25625849

  14. STABLE VARIANTS OF SPERM ANEUPLOIDY AMONG HEALTHY MEN SHOW ASSOCIATIONS BETWEEN GERMINAL AND SOMATIC ANEUPLOIDY

    EPA Science Inventory

    Abstract.

    Our objective was to identify men who consistently produced high frequencies of sperm with numerical chromosomal abnormalities (stable variants) and to determine whether healthy men with normal semen quality vary with respect to the incidence of sperm aneuploidy ...

  15. STABLE VARIANTS OF SPERM ANEUPLOIDY AMONG HEALTHY MEN SHOW ASSOCIATIONS BETWEEN GERMINAL AND SOMATIC ANEUPLOIDY

    EPA Science Inventory

    Stable variants of sperm aneuploidy among healthy men show associations between germinal and somatic aneuploidy

    The purpose of this study was to identify healthy men who reproducibly produced increased frequencies of sperm with numerical chromosomal abnormalities and to d...

  16. How nematode sperm crawl.

    PubMed

    Bottino, Dean; Mogilner, Alexander; Roberts, Tom; Stewart, Murray; Oster, George

    2002-01-15

    Sperm of the nematode, Ascaris suum, crawl using lamellipodial protrusion, adhesion and retraction, a process analogous to the amoeboid motility of other eukaryotic cells. However, rather than employing an actin cytoskeleton to generate locomotion, nematode sperm use the major sperm protein (MSP). Moreover, nematode sperm lack detectable molecular motors or the battery of actin-binding proteins that characterize actin-based motility. The Ascaris system provides a simple 'stripped down' version of a crawling cell in which to examine the basic mechanism of cell locomotion independently of other cellular functions that involve the cytoskeleton. Here we present a mechanochemical analysis of crawling in Ascaris sperm. We construct a finite element model wherein (a) localized filament polymerization and bundling generate the force for lamellipodial extension and (b) energy stored in the gel formed from the filament bundles at the leading edge is subsequently used to produce the contraction that pulls the rear of the cell forward. The model reproduces the major features of crawling sperm and provides a framework in which amoeboid cell motility can be analyzed. Although the model refers primarily to the locomotion of nematode sperm, it has important implications for the mechanics of actin-based cell motility.

  17. Chromatin condensation during terminal erythropoiesis.

    PubMed

    Zhao, Baobing; Yang, Jing; Ji, Peng

    2016-09-02

    Mammalian terminal erythropoiesis involves gradual but dramatic chromatin condensation steps that are essential for cell differentiation. Chromatin and nuclear condensation is followed by a unique enucleation process, which is believed to liberate more spaces for hemoglobin enrichment and enable the generation of a physically flexible mature red blood cell. Although these processes have been known for decades, the mechanisms are still unclear. Our recent study reveals an unexpected nuclear opening formation during mouse terminal erythropoiesis that requires caspase-3 activity. Major histones, except H2AZ, are partially released from the opening, which is important for chromatin condensation. Block of the nuclear opening through caspase inhibitor or knockdown of caspase-3 inhibits chromatin condensation and enucleation. We also demonstrate that nuclear opening and histone release are cell cycle regulated. These studies reveal a novel mechanism for chromatin condensation in mammalia terminal erythropoiesis.

  18. CCSI: a database providing chromatin-chromatin spatial interaction information.

    PubMed

    Xie, Xiaowei; Ma, Wenbin; Songyang, Zhou; Luo, Zhenhua; Huang, Junfeng; Dai, Zhiming; Xiong, Yuanyan

    2016-01-01

    Distal regulatory elements have been shown to regulate gene transcription through spatial interactions, and single nucleotide polymorphisms (SNPs) are linked with distal gene expression by spatial proximity, which helps to explain the causal role of disease-associated SNPs in non-coding region. Therefore, studies on spatial interactions between chromatin have created a new avenue for elucidating the mechanism of transcriptional regulation in disease pathogenesis. Recently, a growing number of chromatin interactions have been revealed by means of 3C, 4C, 5C, ChIA-PET and Hi-C technologies. To interpret and utilize these interactions, we constructed chromatin-chromatin spatial interaction (CCSI) database by integrating and annotating 91 sets of chromatin interaction data derived from published literature, UCSC database and NCBI GEO database, resulting in a total of 3,017,962 pairwise interactions (false discovery rate < 0.05), covering human, mouse and yeast. A web interface has been designed to provide access to the chromatin interactions. The main features of CCSI are (i) showing chromatin interactions and corresponding genes, enhancers and SNPs within the regions in the search page; (ii) offering complete interaction datasets, enhancer and SNP information in the download page; and (iii) providing analysis pipeline for the annotation of interaction data. In conclusion, CCSI will facilitate exploring transcriptional regulatory mechanism in disease pathogenesis associated with spatial interactions among genes, regulatory regions and SNPs. Database URL: http://songyanglab.sysu.edu.cn/ccsi.

  19. Sex chromosome aneuploidies in sperm of 47,XYY men.

    PubMed

    Morel, F; Roux, C; Bresson, J L

    1999-01-01

    The sex chromosomal equipment in 26,675 sperm of 47,XYY males was analyzed. A total of 5.78% of the nuclei exhibited sex chromosome hyperhaploidy. Six studies have analyzed the sperm of 10 XYY patients and, although these studies indicated some degree of elimination of the extra Y chromosome during spermatogenesis, a certain percentage of XYY germinal cells may also be able to achieve meiosis and produce sperm with gonosomal disomies. All these studies show an increased incidence of gonosomal aneuploidies in sperm, but there are significant discrepancies concerning the extent of these abnormalities. The global frequencies of sperm with an abnormal number of sex chromosomes ranged from 0.578 to 13.91%, depending on the patients. There are several explanations for these discrepancies: differences attributed to fluorescence in situ hybridization methodology, the use of dual or multicolor FISH, recruitment, interindividual variations, and intraindividual variations. This study reports an additional series obtained from another XYY individual and compares and discusses the data on gonosomal hyperhaploidies in sperm of 47 XYY males using in situ hybridization analyses.

  20. Functional features and protein network of human sperm-egg interaction.

    PubMed

    Sabetian, Soudabeh; Shamsir, Mohd Shahir; Abu Naser, Mohammed

    2014-12-01

    computationally resolved interactions and the genetic links between sperm-egg interaction abnormalities and the associated disease.

  1. Sperm number trumps sperm size in mammalian ejaculate evolution

    PubMed Central

    Fitzpatrick, John L.

    2015-01-01

    Postcopulatory sexual selection is widely accepted to underlie the extraordinary diversification of sperm morphology. However, why does it favour longer sperm in some taxa but shorter in others? Two recent hypotheses addressing this discrepancy offered contradictory explanations. Under the sperm dilution hypothesis, selection via sperm density in the female reproductive tract favours more but smaller sperm in large, but the reverse in small, species. Conversely, the metabolic constraint hypothesis maintains that ejaculates respond positively to selection in small endothermic animals with high metabolic rates, whereas low metabolic rates constrain their evolution in large species. Here, we resolve this debate by capitalizing on the substantial variation in mammalian body size and reproductive physiology. Evolutionary responses shifted from sperm length to number with increasing mammalian body size, thus supporting the sperm dilution hypothesis. Our findings demonstrate that body-size-mediated trade-offs between sperm size and number can explain the extreme diversification in sperm phenotypes. PMID:26582027

  2. The assessment of sperm DNA fragmentation in the saltwater crocodile (Crocodylus porosus).

    PubMed

    Johnston, S D; López-Fernández, C; Arroyo, F; Fernández, J L; Gosálvez, J

    2015-10-14

    Herein we report a method of assessing DNA fragmentation in the saltwater crocodile using the sperm chromatin dispersion test (SCDt) after including frozen-thawed spermatozoa in a microgel (Halomax; Halotech DNA, Madrid, Spain). Following controlled protein depletion, which included a reducing agent, sperm nuclei with fragmented DNA showed a homogeneous and larger halo of chromatin dispersion with a corresponding reduced nucleoid core compared with sperm with non-fragmented DNA. The presence of DNA damage was confirmed directly by incorporation of modified nucleotides using in situ nick translation (ISNT) and indirectly by studying the correlation of the SCDt with the results of DNA damage visualisation using a two-tailed comet assay (r = 0.90; P = 0.037). Results of the SCDt immediately following thawing and after 5 h incubation at 37°C in order to induce a range of DNA damage revealed individual crocodile differences in both the baseline level of DNA damage and DNA longevity.

  3. Direct visualization of sperm competition and sperm storage in Drosophila.

    PubMed

    Civetta, A

    Drosophila females engage in multiple matings [1] [2] [3] [4] even though they can store sperm in specialized organs for most of their life [5]. The existence of sperm competition in Drosophila has been inferred from the proportion of progeny sired by the second male in double-mating experiments [6] [7] [8]. Investigators have used this approach to quantify genetic variation underlying sperm competition [8] [9] [10], to elucidate its genetic basis [11], to identify the dependence of different male competitive ability on the genotype of the females with which they mate [12] and to discern the potential role of sperm competition in species isolation [13] [14]. This approach assumes that the sperm from two males stored in a female compete to fertilize the eggs. The mechanism by which sperm competition is accomplished is still unknown, however. Here, fluorescence microscopy, cytometry, and differently labeled sperm were used to analyze the fate of sperm inside the female's sperm storage organs, to quantify sperm competition, and to assess how closely paternity success corresponds to the appearance and location of the sperm. The results show that the first male's sperm is retained for a shortened period if the female remates, and that the second males that sire more progeny either induce females to store and use more of their sperm or strongly displace resident sperm.

  4. Intracytoplasmic Sperm Injection (ICSI)

    MedlinePlus

    ... to the inside of the egg (cytoplasm), where fertilization takes place. Sometimes the sperm cannot penetrate the ... ICSI) can be done along with in vitro fertilization (IVF) to help fertilize the egg. During ICSI, ...

  5. Tuning sperm chemotaxis.

    PubMed

    Guerrero, Adán; Wood, Christopher D; Nishigaki, Takuya; Carneiro, Jorge; Darszon, Alberto

    2010-10-01

    Sperm chemotaxis is a long-term puzzle and most of our knowledge comes from studying marine animals that are external fertilizers. Sperm are attracted by diffusible chemical factors (chemoattractants) released from the egg which redirect their swimming paths towards their source. This redirection is driven by increases in flagellar curvature that correlate with transient flagellar Ca(2+) increases. Recent experimental and modelling results provide insights into the signal flow underlying the translation of an external chemical gradient into an intracellular molecular and motor response. A fundamental element of sea-urchin sperm chemotaxis lies in the ability of these cells to suppress Ca(2+)-mediated increases in flagellar curvature while experiencing an increasing chemoattractant gradient. The article considers this new evidence and summarizes the known underlying cellular mechanisms and behavioural strategies that sperm use to locate and fertilize the oocyte.

  6. Single Molecule Studies of Chromatin

    SciTech Connect

    Jeans, C; Colvin, M E; Thelen, M P; Noy, A

    2004-01-06

    The DNA in eukaryotic cells is tightly packaged as chromatin through interactions with histone proteins to form nucleosomes. These nucleosomes are themselves packed together through interactions with linker histone and non-histone proteins. In order for processes such as DNA replication, DNA repair, and transcription to occur, the chromatin fiber must be remodeled such that the necessary enzymes can access the DNA. The structure of the chromatin fiber beyond the level of the single nucleosome and the structural changes which accompany the remodeling process are poorly understood. We are studying the structures and forces behind the remodeling process through the use of atomic force microscopy (AFM). This allows both high-resolution imaging of the chromatin, and manipulation of individual fibers. Pulling a single chromatin fiber apart using the AFM tip yields information on the forces which hold the structure together. We have isolated chromatin fibers from chicken erythrocytes and Chinese hamster ovary cell lines. AFM images of these fibers will be presented, along with preliminary data from the manipulation of these fibers using the AFM tip. The implications of these data for the structure of chromatin undergoing the remodeling process are discussed.

  7. Chromatin and Transcription in Yeast

    PubMed Central

    Rando, Oliver J.; Winston, Fred

    2012-01-01

    Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field. PMID:22345607

  8. Mesoscale Modeling of Chromatin Folding

    NASA Astrophysics Data System (ADS)

    Schlick, Tamar

    2009-03-01

    Eukaryotic chromatin is the fundamental protein/nucleic acid unit that stores the genetic material. Understanding how chromatin fibers fold and unfold in physiological conditions is important for interpreting fundamental biological processes like DNA replication and transcription regulation. Using a mesoscopic model of oligonucleosome chains and tailored sampling protocols, we elucidate the energetics of oligonucleosome folding/unfolding and the role of each histone tail, linker histones, and divalent ions in regulating chromatin structure. The resulting compact topologies reconcile features of the zigzag model with straight linker DNAs with the solenoid model with bent linker DNAs for optimal fiber organization and reveal dynamic and energetic aspects involved.

  9. Effects of centrifugation through three different discontinuous Percoll gradients on boar sperm function.

    PubMed

    Matás, C; Vieira, L; García-Vázquez, F A; Avilés-López, K; López-Úbeda, R; Carvajal, J A; Gadea, J

    2011-08-01

    In this study, different combinations of 2-step, discontinuous gradient centrifugation were used, consisting of three different combinations of isotonic Percoll (45/60, 60/75 and 45/90%) that allowed us to select different sperm subpopulations from fertile and normozoospermic boars. Our objective in this study is to evaluate the effects of centrifugation through three different discontinuous Percoll gradients on sperm function parameters (motility, viability, morphology, acrosome status, chromatin condensation, DNA fragmentation, ROS generation, tyrosine phosphorylation and intracellular calcium concentration) and the sperm penetrating capacity in an IVF system. All the Percoll treatments evaluated increased the percentage of spermatozoa with normal morphology, the proportion of un-damaged DNA, normal chromatin condensation, motion parameters measured by CASA and the percentage of capacitated spermatozoa with tyrosine phosphorylated proteins compared to control group. Finally, the in vitro oocyte penetrating capacity of boar spermatozoa was significantly affected by Percoll centrifugation. All the Percoll treatments increased the penetration rates and mean number of sperm per penetrated oocyte. Despite the efficiency of all three of the sperm treatments tested in selecting spermatozoa with improved sperm parameters and capacity to penetrate oocytes in vitro, the optimum performance of this system was demonstrated after preselecting spermatozoa by centrifugation on a discontinuous 45/90 Percoll gradient. The P45/90 treatment leads to obtain a higher percentage of spermatozoa which develop properly the capacitation process as it was shown measuring tyrosine phosphorylation and intracellular calcium concentration.

  10. Sperm DNA Fragmentation Index and Hyaluronan Binding Ability in Men from Infertile Couples and Men with Testicular Germ Cell Tumor.

    PubMed

    Marchlewska, Katarzyna; Filipiak, Eliza; Walczak-Jedrzejowska, Renata; Oszukowska, Elzbieta; Sobkiewicz, Slawomir; Wojt, Malgorzata; Chmiel, Jacek; Kula, Krzysztof; Slowikowska-Hilczer, Jolanta

    2016-01-01

    Objective. To investigate sperm DNA fragmentation and sperm functional maturity in men from infertile couples (IC) and men with testicular germ cell tumor (TGCT). Materials and Methods. Semen samples were collected from 312 IC men and 23 men with TGCT before unilateral orchiectomy and oncological treatment. The sperm chromatin dispersion test was performed to determine DNA fragmentation index (DFI) and the ability of sperm to bind with hyaluronan (HA) was assessed. Results. In comparison with the IC men, the men with TGCT had a higher percentage of sperm with fragmented DNA (median 28% versus 21%; p < 0.01) and a lower percentage of HA-bound sperm (24% versus 66%; p < 0.001). Normal results of both analyses were observed in 24% of IC men and 4% of men with TGCT. Negative Spearman's correlations were found between DFI and the percentage of HA-bound sperm in the whole group and in IC subjects and those with TGCT analyzed separately. Conclusions. Approximately 76% of IC men and 96% with TGCT awaiting orchiectomy demonstrated DNA fragmentation and/or sperm immaturity. We therefore recommend sperm banking after unilateral orchiectomy, but before irradiation and chemotherapy; the use of such a deposit appears to be a better strategy to obtain functionally efficient sperms.

  11. Sperm DNA Fragmentation Index and Hyaluronan Binding Ability in Men from Infertile Couples and Men with Testicular Germ Cell Tumor

    PubMed Central

    Filipiak, Eliza; Walczak-Jedrzejowska, Renata; Oszukowska, Elzbieta; Sobkiewicz, Slawomir; Wojt, Malgorzata; Chmiel, Jacek; Kula, Krzysztof; Slowikowska-Hilczer, Jolanta

    2016-01-01

    Objective. To investigate sperm DNA fragmentation and sperm functional maturity in men from infertile couples (IC) and men with testicular germ cell tumor (TGCT). Materials and Methods. Semen samples were collected from 312 IC men and 23 men with TGCT before unilateral orchiectomy and oncological treatment. The sperm chromatin dispersion test was performed to determine DNA fragmentation index (DFI) and the ability of sperm to bind with hyaluronan (HA) was assessed. Results. In comparison with the IC men, the men with TGCT had a higher percentage of sperm with fragmented DNA (median 28% versus 21%; p < 0.01) and a lower percentage of HA-bound sperm (24% versus 66%; p < 0.001). Normal results of both analyses were observed in 24% of IC men and 4% of men with TGCT. Negative Spearman's correlations were found between DFI and the percentage of HA-bound sperm in the whole group and in IC subjects and those with TGCT analyzed separately. Conclusions. Approximately 76% of IC men and 96% with TGCT awaiting orchiectomy demonstrated DNA fragmentation and/or sperm immaturity. We therefore recommend sperm banking after unilateral orchiectomy, but before irradiation and chemotherapy; the use of such a deposit appears to be a better strategy to obtain functionally efficient sperms. PMID:27999814

  12. Correlation between sperm DNA fragmentation index and CMA3 positive spermatozoa in globozoospermic patients.

    PubMed

    Hosseinifar, H; Yazdanikhah, S; Modarresi, T; Totonchi, M; Sadighi Gilani, M A; Sabbaghian, M

    2015-05-01

    The absence of the acrosome causes the situation which is called globozoospermia. There are a few studies, mostly as case reports, about correlation between levels of sperm DNA damage in patients with total round-headed spermatozoa. We investigated this correlation as well as CMA3 positive spermatozoa in 20 globozoospermic men (with more than 90% round-headed spermatozoa) attending to Royan Institute. Semen samples divided into three parts to semen analysis, to measure DNA fragmentation index (DFI) using sperm chromatin structure assay (SCSA) and to detect CMA3(+) sperm cells by chromomycin A3 staining and fluorescent microscopy. Our results showed that there were significant differences in sperm concentration, total sperm motility, and normal morphology between patients and controls group (p < 0.001). Moreover, the average of DFI and CMA3 positive spermatozoa in patients group significantly increases compared with control group (p < 0.001). A significant correlation between DFI and CMA3(+) in total population was also detected in patients group (r = 0.45, p = 0.046). To our knowledge, this is the largest study about correlation between DNA damage levels and CMA3 positive spermatozoa with round head sperm cells in total globozoospermic men. It seems that the increase in DNA damage may be because of defective sperm DNA compaction, as we detected CMA3 positive sperm cells in these patients.

  13. Initial analysis of sperm DNA methylome in Holstein bulls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aberrant DNA methylation patterns have been associated with abnormal semen parameters, idiopathic male infertility and early embryonic loss in mammals. Using Holstein bulls with high (Bull1) or low (Bull2) fertility rates, we created two representative sperm DNA methylomes at a single-base resolutio...

  14. Unusual chromatin in human telomeres.

    PubMed Central

    Tommerup, H; Dousmanis, A; de Lange, T

    1994-01-01

    We report that human telomeres have an unusual chromatin structure characterized by diffuse micrococcal nuclease patterns. The altered chromatin manifested itself only in human telomeres that are relatively short (2 to 7 kb). In contrast, human and mouse telomeres with telomeric repeat arrays of 14 to 150 kb displayed a more canonical chromatin structure with extensive arrays of tightly packed nucleosomes. All telomeric nucleosomes showed a shorter repeat size than bulk nucleosomes, and telomeric mononucleosomal particles were found to be hypersensitive to micrococcal nuclease. However, telomeric nucleosomes were similar to bulk nucleosomes in the rate at which they sedimented through sucrose gradients. We speculate that mammalian telomeres have a bipartite structure with unusual chromatin near the telomere terminus and a more canonical nucleosomal organization in the proximal part of the telomere. Images PMID:8065312

  15. Regulation of cellular chromatin state

    PubMed Central

    Mishra, Rakesh K; Dhawan, Jyotsna

    2010-01-01

    The identity and functionality of eukaryotic cells is defined not just by their genomic sequence which remains constant between cell types, but by their gene expression profiles governed by epigenetic mechanisms. Epigenetic controls maintain and change the chromatin state throughout development, as exemplified by the setting up of cellular memory for the regulation and maintenance of homeotic genes in proliferating progenitors during embryonic development. Higher order chromatin structure in reversibly arrested adult stem cells also involves epigenetic regulation and in this review we highlight common trends governing chromatin states, focusing on quiescence and differentiation during myogenesis. Together, these diverse developmental modules reveal the dynamic nature of chromatin regulation providing fresh insights into the role of epigenetic mechanisms in potentiating development and differentiation. PMID:20592864

  16. The micronutrient supplements, zinc sulphate and folic acid, did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men.

    PubMed

    Raigani, M; Yaghmaei, B; Amirjannti, N; Lakpour, N; Akhondi, M M; Zeraati, H; Hajihosseinal, M; Sadeghi, M R

    2014-01-01

    We investigated the effects of folic acid and zinc sulphate supplementation on the improvement of sperm function in subfertile oligoasthenoteratozoospermic (OAT) men. Eighty-three OAT men participated in a 16-week intervention randomised, double-blind clinical trial with daily treatment of folic acid (5 mg day(-1) ) and zinc sulphate (220 mg day(-1) ), or placebo. Before and after treatment, semen and blood samples were obtained for determining sperm concentration, motility, and morphology, sperm viability, sperm mitochondrial function, sperm chromatin status using toluidine blue, aniline blue, acridine orange and chromomycin A3 staining; and semen and blood folate, zinc, B12 , total antioxidant capacity (TAC) and malondialdehyde (MDA) concentrations. Sperm concentration (×10(6)  ml(-1) ) increased in subfertile men receiving the combined treatment of folic acid and zinc sulphate and also in the group receiving only folic acid treatment; however, it was not statistically significant (P = 0.056 and P = 0.05, respectively). Sperm chromatin integrity (%) increased significantly in subfertile men receiving only zinc sulphate treatment (P = 0.048). However, this improvement in sperm quality was not significant after adjusting placebo effect. This study showed that zinc sulphate and folic acid supplementation did not ameliorate sperm quality in infertile men with severely compromised sperm parameters, OAT. Male infertility is a multifactorial disorder, and also nutritional factors play an important role in results of administration of supplementation on sperm parameters. However, these results should be confirmed by multiple studies in larger populations of OAT men.

  17. Quantification of mammalian sperm morphology by slit-scan flow cytometry

    SciTech Connect

    Benaron, D.A.; Gray, J.W.; Gledhill, B.L.; Lake, S.; Wyrobek, A.J.; Young, I.T.

    1982-01-01

    The head shapes of mammalian sperm were measured by slit-scan flow cytometry (SSFCM). Fluorescence profiles were measured for sperm from mice, rabbits, hamsters, and bulls, and for sperm from mice, rabbits, hamsters, and bulls, and for sperm from mice exposed to testicular x-irradiation from 0 to 900 rads. Some of the fluorescence profiles for sperm from the irradiated mice differed significantly from the profiles usually measured for sperm from unexposed mice. An algorithm was developed to determine the frequency of these sperm. The estimated frequencies of atypical profiles correlated well with the frequencies of abnormally shaped sperm determined by microscopic scoring. The maximum SSFCM sensitivity was not as high as that for the visual assay. However, only 100 profiles were measured by SSFCM at each dose while at least 500 sperm were scored visually at each dose. The sensitivity of the SSFCM assay should be increased substantially by measuring more profiles. The objective nature of SSFCM coupled with the high correlation with results from the visually based assay of morphology suggests the use of SSFCM to measure frequencies of misshapen sperm when testing for mutagens or monitoring for effects of environmental contaminants.

  18. Sperm Motility in Flow

    NASA Astrophysics Data System (ADS)

    Guasto, Jeffrey; Juarez, Gabriel; Stocker, Roman

    2012-11-01

    A wide variety of plants and animals reproduce sexually by releasing motile sperm that seek out a conspecific egg, for example in the reproductive tract for mammals or in the water column for externally fertilizing organisms. Sperm are aided in their quest by chemical cues, but must also contend with hydrodynamic forces, resulting from laminar flows in reproductive tracts or turbulence in aquatic habitats. To understand how velocity gradients affect motility, we subjected swimming sperm to a range of highly-controlled straining flows using a cross-flow microfluidic device. The motion of the cell body and flagellum were captured through high-speed video microscopy. The effects of flow on swimming are twofold. For moderate velocity gradients, flow simply advects and reorients cells, quenching their ability to cross streamlines. For high velocity gradients, fluid stresses hinder the internal bending of the flagellum, directly inhibiting motility. The transition between the two regimes is governed by the Sperm number, which compares the external viscous stresses with the internal elastic stresses. Ultimately, unraveling the role of flow in sperm motility will lead to a better understanding of population dynamics among aquatic organisms and infertility problems in humans.

  19. Sperm-egg interaction.

    PubMed

    Evans, Janice P

    2012-01-01

    A crucial step of fertilization is the sperm-egg interaction that allows the two gametes to fuse and create the zygote. In the mouse, CD9 on the egg and IZUMO1 on the sperm stand out as critical players, as Cd9(-/-) and Izumo1(-/-) mice are healthy but infertile or severely subfertile due to defective sperm-egg interaction. Moreover, work on several nonmammalian organisms has identified some of the most intriguing candidates implicated in sperm-egg interaction. Understanding of gamete membrane interactions is advancing through characterization of in vivo and in vitro fertilization phenotypes, including insights from less robust phenotypes that highlight potential supporting (albeit not absolutely essential) players. An emerging theme is that there are varied roles for gamete molecules that participate in sperm-egg interactions. Such roles include not only functioning as fusogens, or as adhesion molecules for the opposite gamete, but also functioning through interactions in cis with other proteins to regulate membrane order and functionality.

  20. Healthy Sperm: Improving Your Fertility

    MedlinePlus

    Healthy Lifestyle Getting pregnant Healthy sperm aren't always a given. Understand how lifestyle factors can affect your ... as a laptop, might enhance sperm quality. Adopting healthy lifestyle practices to promote your fertility — and avoiding things ...

  1. Functional studies of MP62 during male chromatin decondensation in sea urchins.

    PubMed

    Iribarren, Claudio; Hermosilla, Viviana; Morin, Violeta; Puchi, Marcia

    2013-08-01

    In amphibians, sperm histone transition post-fertilization during male pronucleus formation is commanded by histone chaperone Nucleoplasmin (NPM). Here, we report the first studies to analyze the participation of a Nucleoplasmin-like protein on male chromatin remodeling in sea urchins. In this report, we present the molecular characterization of a nucleoplasmin-like protein that is present in non fertilized eggs and early zygotes in sea urchin specie Tetrapygus niger. This protein, named MP62 can interact with sperm histones in vitro. By male chromatin decondensation assays and immunodepletion experiments in vitro, we have demonstrated that this protein is responsible for sperm nucleosome disorganization. Furthermore, as amphibian nucleoplasmin MP62 is phosphorylated in vivo immediately post-fertilization and this phosphorylation is dependent on CDK-cyclin activities found after fertilization. As we shown, olomoucine and roscovitine inhibits male nucleosome decondensation, sperm histone replacement in vitro and MP62 phosphorylation in vivo. This is the first report of a nucleoplasmin-like activity in sea urchins participating during male pronucleus formation post-fecundation.

  2. Dynamic aspects of spermiogenic chromatin condensation patterning by phase separation during the histone-to-protamine transition in charalean algae and relation to bryophytes.

    PubMed

    Kasinsky, H E; Ellis, S; Martens, G; Ausió, J

    2014-12-01

    During early-to-middle spermiogenesis in multicellular, internally fertilizing charalean green algae (Chara fibrosa, Chara vulgaris, Chara tomentosa, Nitella missouriensis), patterning of chromatin/nucleoplasm in developing spermatid nuclei changes from granules → fibers → contorted lamellae → condensed chromatin. Cytochemical, immunocytochemical, electrophoretic studies on C. vulgaris and C. tomentosa spermatids (Kwiatkowska, Poplonska) and amino acid analysis of protamines in Chara corallina sperm (Reynolds, Wolfe), indicate that more positively charged protamines replace histones directly during spermiogenesis, not indirectly through other intermediate transitional proteins as in internally fertilizing neogastropods and sharks with more ordered spermatid lamellae. We hypothesize that such lamellar-mediated patterning is due to liquid-liquid phase separation by spinodal decomposition. This is a spontaneous thermodynamic process that involves diffusive instability of a lamellar chromatin network, a dominant pattern repeat distance and bicontinuity of chromatin/nucleoplasm phases. C. vulgaris sperm show contorted lamellae in the posterior region, whereas C. corallina sperm display contorted peripheral lamellae and interior fibrils. Among internally fertilizing liverworts, which may have evolved from Zygnematales, mid-spermatid nuclei lack lamellae. Instead they display self-coiled chromatin rods in Blasia pusilla, contain short chromatin tubules in Haplomitrium hookeri resembling those in internally fertilizing mosses and a hornwort and indirectly replace histones with protamines in Marchantia polymorpha.

  3. Exposure to Endosulfan can result in male infertility due to testicular atrophy and reduced sperm count

    PubMed Central

    Sebastian, R; Raghavan, SC

    2015-01-01

    Endosulfan (ES) is a widely used organochlorine pesticide and is speculated to be detrimental to human health. However, very little is known about mechanism of its genotoxicity. Using mouse model system, we show that exposure to ES affected physiology and cellular architecture of organs and tissues. Among all organs, damage to testes was extensive and it resulted in death of different testicular-cell populations. We find that the damage in testes resulted in qualitative and quantitative defects during spermatogenesis in a time-dependent manner, increasing epididymal reactive oxygen species levels, affecting sperm chromatin integrity. This further culminated in reduced number of epididymal sperms and actively motile sperms. Finally, we show that ES exposure affected fertility in male but not in female mice. Therefore, we demonstrate that ES exerts pathophysiological changes in mice, induces testicular atrophy, affects spermatogenesis, reduces quantity and vigour of epididymal sperm and leads to infertility in males. PMID:27551453

  4. Differential clustering of sperm subpopulations in infertile males with clinical varicocele and carriers of rearranged genomes.

    PubMed

    García-Peiró, Agustín; Oliver-Bonet, María; Navarro, Joaquima; Abad, Carlos; Amengual, María José; López-Fernández, Carmen; Gosálvez, Jaime; Benet, Jordi

    2012-01-01

    Some methods for determining sperm DNA fragmentation, such as the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion test (SCD), provide additional information about particular subgroups of spermatozoa with specific irregularities. Thus, SCSA recognizes a specific sperm subpopulation, the high-DNA stainability sperm subpopulation (HDS), and SCD recognizes the so-called DNA-degraded sperm (DDS) subpopulation. Although some studies associate the presence of these subpopulations with specific aspects related to infertility, the relationship between both sperm subpopulations and their preponderance in specific clinical groups of infertile males has not been extensively investigated. In this study, HDS and DDS subpopulations were determined in a total of 37 human males: 8 males with proven fertility, 9 infertile males with asthenoteratozoospermia, 10 carriers of chromosomal reorganizations, and 10 infertile males with clinical varicocele. Results showed a significant increase of the DDS subpopulation (P < .001) in both the varicocele patient (16.85 ± 7.24) and carrier of rearranged genome (11.6 ± 5.23) groups, but not in patients with asthenoteratozoospermia (3.88 ± 1.55) or fertile donors (2.62 ± 1.68). No statistical differences were detected for the HDS subpopulation (P = .542), but the highest values were found in the varicocele and rearranged-genome groups. However, no correlation between the HDS and DDS subpopulations were found (r = 0.196; P = .244), suggesting that both represent a different class of sperm subpopulation in the ejaculate. A significant increase in HDS, and especially DDS, can be associated with the presence of varicocele or the rearrangement of chromosomes. Specific diagnostic tests to confirm the diagnosis must be performed in patients with increased DDS and HDS values.

  5. Radiation-induced DNA content variability in mouse sperm

    SciTech Connect

    Pinkel, D.; Gledhill, B.L.; van Dilla, M.A.; Lake, S.; Wyrobek, A.J.

    1983-09-01

    Mouse sperm collected from the cauda epididymidis 35 days after acute testicular x-ray exposure and fluorescently stained for DNA show dose-dependent increases in the coefficient of variation (CV) of flow cytometrically obtained fluorescence distributions. By comparing dose-response curves obtained with three protocols which overcome the optical and cytochemical difficulties of sperm measurement in different ways we conclude the response is due to x-ray-induced DNA content variability. Computer modeling of the shapes of the fluorescence distributions show that at 600 rad 30 to 40% of the sperm have abnormal DNA content. Some have errors as large as two whole chromosomes, but it is not clear whether they are due to whole chromosome nondisjunction or a finer fragmentation of the genome. Exposures to benzo(a)pyrene and mitomycin C cause no detectable DNA content variability. We conclude mouse sperm DNA content measurements are not sensitive to small amounts of aneuploidy and as such will only be useful in detecting agents that produce substantial DNA content variability. Another animal with a smaller number of chromosomes might be more favorable. These sperm measurement techniques may find additional application in other areas of reproductive biology, such as the determination of the relative numbers of X and Y chromosome-bearing sperm in semen that may be artifically enriched in one population.

  6. Leukocyte abnormalities.

    PubMed

    Gabig, T G

    1980-07-01

    Certain qualitative abnormalities in neutrophils and blood monocytes are associated with frequent, severe, and recurrent bacterial infections leading to fatal sepsis, while other qualitative defects demonstrated in vitro may have few or no clinical sequelae. These qualitative defects are discussed in terms of the specific functions of locomotion, phagocytosis, degranulation, and bacterial killing.

  7. Individual adjustment of sperm expenditure accords with sperm competition theory.

    PubMed

    Pilastro, Andrea; Scaggiante, Marta; Rasotto, Maria B

    2002-07-23

    Sperm competition theory predicts that males should strategically allocate their sperm reserves according to the level of sperm competition, defined as the probability that the sperm of two males compete for fertilizing a given set of ova. Substantial evidence from numerous animal taxa suggests that, at the individual level, sperm expenditure increases when the risk of sperm competition is greater. In contrast, according to the "intensity model" of sperm competition [Parker, G. A., Ball, M. A., Stockley, P. & Gage, M. J. G. (1996) Proc. R. Soc. London Ser. B 263, 1291-1297], when more than two ejaculates compete during a given mating event, sperm expenditure should decrease as the number of competing males increases. Empirical evidence supporting this prediction, however, is still lacking. Here we measured sperm expenditure in two gobiid fishes, the grass (Zosterisessor ophiocephalus) and black goby (Gobius niger), in which up to six sneakers can congregate around the nest of territorial males and release their sperm when females spawn. We show that, in accordance with theory, sneaker males of both species release fewer sperm as the number of competitors increases.

  8. Chromatin structure and DNA damage

    SciTech Connect

    Gale, J.M.

    1987-01-01

    This dissertation examines the structure and structural transitions of chromatin in relation to DNA damage. The ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro was examined following DNA photodamage introduced by two different agents. (1) 254-nm UV radiation and (2) trimethylpsoralen (plus near-UV radiation). Both agents are highly specific for DNA and form adducts predicted to cause different degrees of distortion in the DNA helix. The salt-induced structural transitions of intact and histone H1 depleted chromatin fibers were monitored by both analytical ultracentrifugation and light scattering. Our results show that even in the presence of extremely large, nonphysiological amounts of photodamage by either agent the ability of chromatin to fold into higher ordered structures is not affected. The compact, 30 nm fiber must therefore be able to accommodate a large amount of DNA damage without any measurable changes in the overall size or degree of compaction of this structure. The distribution of pyrimidine dimers was mapped at the single nucleotide level in nucleosome core DNA from UV-irradiated mononucleosomes, chromatin fibers, and human cells in culture using the 3' ..-->.. 5' exonuclease activity of T4 DNA polymerase.

  9. Chromatin modification in zebrafish development.

    PubMed

    Cayuso Mas, Jordi; Noël, Emily S; Ober, Elke A

    2011-01-01

    The generation of complex organisms requires that an initial population of cells with identical gene expression profiles can adopt different cell fates during development by progressively diverging transcriptional programs. These programs depend on the binding of transcritional regulators to specific genomic sites, which in turn is controlled by modifications of the chromatin. Chromatin modifications may occur directly upon DNA by methylation of specific nucleotides, or may involve post-translational modification of histones. Local regulation of histone post-translational modifications regionalizes the genome into euchromatic regions, which are more accessible to DNA-binding factors, and condensed heterochromatic regions, inhibiting the binding of such factors. In addition, these modifications may be required in a genome-wide fashion for processes such as DNA replication or chromosome condensation. From an embryologist's point of view chromatin modifications are intensively studied in the context of imprinting and have more recently received increasing attention in understanding the basis of pluripotency and cellular differentiation. Here, we describe recently uncovered roles of chromatin modifications in zebrafish development and regeneration, as well as available resources and commonly used techniques. We provide a general introduction into chromatin modifications and their respective functions with a focus on gene transcription, as well as key aspects of their roles in the early zebrafish embryo, neural development, formation of the digestive system and tissue regeneration.

  10. Chromatin shapes the mitotic spindle.

    PubMed

    Dinarina, Ana; Pugieux, Céline; Corral, Maria Mora; Loose, Martin; Spatz, Joachim; Karsenti, Eric; Nédélec, François

    2009-08-07

    In animal and plant cells, mitotic chromatin locally generates microtubules that self-organize into a mitotic spindle, and its dimensions and bipolar symmetry are essential for accurate chromosome segregation. By immobilizing microscopic chromatin-coated beads on slide surfaces using a microprinting technique, we have examined the effect of chromatin on the dimensions and symmetry of spindles in Xenopus laevis cytoplasmic extracts. While circular spots with diameters around 14-18 microm trigger bipolar spindle formation, larger spots generate an incorrect number of poles. We also examined lines of chromatin with various dimensions. Their length determined the number of poles that formed, with a 6 x 18 microm rectangular patch generating normal spindle morphology. Around longer lines, multiple poles formed and the structures were disorganized. While lines thinner than 10 mum generated symmetric structures, thicker lines induced the formation of asymmetric structures where all microtubules are on the same side of the line. Our results show that chromatin defines spindle shape and orientation. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.

  11. Single Molecule Studies of Chromatin

    SciTech Connect

    Jeans, C; Thelen, M P; Noy, A

    2006-02-06

    In eukaryotic cells, DNA is packaged as chromatin, a highly ordered structure formed through the wrapping of the DNA around histone proteins, and further packed through interactions with a number of other proteins. In order for processes such as DNA replication, DNA repair, and transcription to occur, the structure of chromatin must be remodeled such that the necessary enzymes can access the DNA. A number of remodeling enzymes have been described, but our understanding of the remodeling process is hindered by a lack of knowledge of the fine structure of chromatin, and how this structure is modulated in the living cell. We have carried out single molecule experiments using atomic force microscopy (AFM) to study the packaging arrangements in chromatin from a variety of cell types. Comparison of the structures observed reveals differences which can be explained in terms of the cell type and its transcriptional activity. During the course of this project, sample preparation and AFM techniques were developed and optimized. Several opportunities for follow-up work are outlined which could provide further insight into the dynamic structural rearrangements of chromatin.

  12. Quantification of leptin in seminal plasma of buffalo bulls and its correlation with antioxidant status, conventional and computer-assisted sperm analysis (CASA) semen variables.

    PubMed

    Kumar, Pradeep; Saini, Monika; Kumar, Dharmendra; Jan, M H; Swami, Dheer Singh; Sharma, R K

    2016-03-01

    The present study is the first to quantify leptin in seminal plasma of buffalo and investigate its relationship with seminal attributes. Ten ejaculates each from 10 Murrah buffalo bulls were collected. Semen quality variables such as semen volume, sperm concentration, sperm abnormalities, membrane integrity, antioxidant enzyme activities (superoxide dismutase, catalase and total antioxidant capacity), malondialdehyde (MDA) concentration, as well as sperm kinetics and motility variables were evaluated. The leptin concentration in serum and seminal plasma were estimated by the ELISA method. Bulls were classified in two groups on the basis of sperm concentration with Group I having >800 million sperm/mL and Group II <500 million sperm/mL. Greater (P<0.05) mean sperm abnormalities, seminal leptin concentrations and MDA concentrations were recorded in Group II than Group I. The seminal leptin was positively correlated with sperm abnormalities and MDA concentration while being negatively correlated with sperm concentration, but there was no correlation with sperm kinetic and motility variables, sperm membrane integrity and seminal plasma antioxidant enzyme activity. Thus, the data suggest that seminal leptin has a role in spermatogenesis and can be used as a marker for spermatogenesis to predict the capacity of buffalo bulls for semen production.

  13. Chromatin Remodeling and Plant Immunity.

    PubMed

    Chen, W; Zhu, Q; Liu, Y; Zhang, Q

    2017-01-01

    Chromatin remodeling, an important facet of the regulation of gene expression in eukaryotes, is performed by two major types of multisubunit complexes, covalent histone- or DNA-modifying complexes, and ATP-dependent chromosome remodeling complexes. Snf2 family DNA-dependent ATPases constitute the catalytic subunits of ATP-dependent chromosome remodeling complexes, which accounts for energy supply during chromatin remodeling. Increasing evidence indicates a critical role of chromatin remodeling in the establishment of long-lasting, even transgenerational immune memory in plants, which is supported by the findings that DNA methylation, histone deacetylation, and histone methylation can prime the promoters of immune-related genes required for disease defense. So what are the links between Snf2-mediated ATP-dependent chromosome remodeling and plant immunity, and what mechanisms might support its involvement in disease resistance?

  14. Sex aneuploidy of unfertilized human oocytes after intracytoplasmic sperm injection

    SciTech Connect

    Lee, G.; Ward, D.C.; Jones, E.E.

    1994-09-01

    Intracytoplasmic sperm injection (ICSI) has recently achieved successful fertilization and pregnancy in human in vitro fertilization, particularly in cases of severe male factor infertility. One criticism of this novel clinical technique is that it bypasses the natural selection process of fertilization. We use fluorescence in situ hybridization (FISH) to analyze oocytes which fail to fertilize after ICSI in the Yale IVF Program. The purpose of this study is to determine whether failed fertilization after ICSI can be attributed to sex chromosome aneuploidy in the oocyte. Fertilization of oocytes is determined by the presence of two pronuclei on light microscopic examination (400X). Multi-probe FISH with DAPI (4,6,-diamino-2-phenyl-indole) counterstain is then performed to determine oocyte ploidy and the presence of decondensed sperm. Centromeric probes for X, Y and 17 are used simultaneously in each oocyte for in situ hybridization to oocyte chromatin. In all oocytes examined after ICSI to date, unfertilized oocytes have decondensed sperm DNA present confirming appropriate intracytoplasmic placement of the sperm. Preliminary results obtained from 31 oocytes have not identified any sex chromosome aneuploidies. The FISH technique used in post-ICSI oocytes is a model system for delineating genetic causes of failed fertilization in the human.

  15. Iodixanol density gradient centrifugation for selecting stallion sperm for cold storage and cryopreservation.

    PubMed

    Stuhtmann, Gesa; Oldenhof, Harriëtte; Peters, Pamela; Klewitz, Jutta; Martinsson, Gunilla; Sieme, Harald

    2012-08-01

    Density gradient centrifugation can be used for selection of sperm of superior quality and removal of seminal plasma for use in artificial insemination. In this study, the use of two-layer iodixanol density gradient centrifugation was evaluated for processing of stallion semen. The protocol includes centrifugation through a 16% iodixanol top layer of 1.090 g mL(-1) and collection of motile and intact sperm on a 30% iodixanol bottom layer of 1.165 g mL(-1). Sperm recovery and effects on sperm quality were determined during cold storage as well as after cryopreservation and compared with ordinary dilution and centrifugation. Two-layer iodixanol density gradient centrifugation allows for selection of greater percentages of morphologically normal and progressively motile sperm compared to ordinary centrifugation. This likely results from collecting sperm on the bottom layer that functions as cushion fluid, which prevents mechanical forces as occur when sperm are packed in a pellet. In addition, percentages of membrane and chromatin integrity are increased when cells are selected based on their density via centrifugation through the top and bottom layers. Removal of seminal plasma and increased initial percentages of motile and membrane intact sperm after iodixanol density gradient centrifugation also result in greater percentages of progressively motile and membrane intact sperm during cold storage as well as after freezing and thawing. In conclusion, the two-layer iodixanol density gradient centrifugation protocol described in this manuscript allows for selection of stallion sperm with greater survival rates for cold storage and cryopreservation.

  16. Phosphoglycerate Kinase 2 (PGK2) Is Essential for Sperm Function and Male Fertility in Mice1

    PubMed Central

    Danshina, Polina V.; Geyer, Christopher B.; Dai, Qunsheng; Goulding, Eugenia H.; Willis, William D.; Kitto, G. Barrie; McCarrey, John R.; Eddy, E.M.; O'Brien, Deborah A.

    2010-01-01

    Phosphoglycerate kinase 2 (PGK2), an isozyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by an autosomal retrogene that is expressed only during spermatogenesis. It replaces the ubiquitously expressed phosphoglycerate kinase 1 (PGK1) isozyme following repression of Pgk1 transcription by meiotic sex chromosome inactivation during meiotic prophase and by postmeiotic sex chromatin during spermiogenesis. The targeted disruption of Pgk2 by homologous recombination eliminates PGK activity in sperm and severely impairs male fertility, but does not block spermatogenesis. Mating behavior, reproductive organ weights (testis, excurrent ducts, and seminal vesicles), testis histology, sperm counts, and sperm ultrastructure were indistinguishable between Pgk2−/− and wild-type mice. However, sperm motility and ATP levels were markedly reduced in males lacking PGK2. These defects in sperm function were slightly less severe than observed in males lacking glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS), the isozyme that catalyzes the step preceding PGK2 in the sperm glycolytic pathway. Unlike Gapdhs−/− males, the Pgk2−/− males also sired occasional pups. Alternative pathways that bypass the PGK step of glycolysis exist. We determined that one of these bypass enzymes, acylphosphatase, is active in mouse sperm, perhaps contributing to phenotypic differences between mice lacking GAPDHS or PGK2. This study determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility. In addition to confirming the importance of the glycolytic pathway for sperm function, distinctive phenotypic characteristics of Pgk2−/− mice may provide further insights into the regulation of sperm metabolism. PMID:19759366

  17. Phosphoglycerate kinase 2 (PGK2) is essential for sperm function and male fertility in mice.

    PubMed

    Danshina, Polina V; Geyer, Christopher B; Dai, Qunsheng; Goulding, Eugenia H; Willis, William D; Kitto, G Barrie; McCarrey, John R; Eddy, E M; O'Brien, Deborah A

    2010-01-01

    Phosphoglycerate kinase 2 (PGK2), an isozyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by an autosomal retrogene that is expressed only during spermatogenesis. It replaces the ubiquitously expressed phosphoglycerate kinase 1 (PGK1) isozyme following repression of Pgk1 transcription by meiotic sex chromosome inactivation during meiotic prophase and by postmeiotic sex chromatin during spermiogenesis. The targeted disruption of Pgk2 by homologous recombination eliminates PGK activity in sperm and severely impairs male fertility, but does not block spermatogenesis. Mating behavior, reproductive organ weights (testis, excurrent ducts, and seminal vesicles), testis histology, sperm counts, and sperm ultrastructure were indistinguishable between Pgk2(-/-) and wild-type mice. However, sperm motility and ATP levels were markedly reduced in males lacking PGK2. These defects in sperm function were slightly less severe than observed in males lacking glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS), the isozyme that catalyzes the step preceding PGK2 in the sperm glycolytic pathway. Unlike Gapdhs(-/-) males, the Pgk2(-/-) males also sired occasional pups. Alternative pathways that bypass the PGK step of glycolysis exist. We determined that one of these bypass enzymes, acylphosphatase, is active in mouse sperm, perhaps contributing to phenotypic differences between mice lacking GAPDHS or PGK2. This study determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility. In addition to confirming the importance of the glycolytic pathway for sperm function, distinctive phenotypic characteristics of Pgk2(-/-) mice may provide further insights into the regulation of sperm metabolism.

  18. Comparison among different cryoprotectants for cryopreservation of epididymal sperm from agouti (Dasyprocta leporina).

    PubMed

    Castelo, T S; Silva, A M; Bezerra, L G P; Costa, C Y M; Lago, A E A; Bezerra, J A B; Campos, L B; Praxedes, E C G; Silva, A R

    2015-12-01

    We verify the effects of different cryoprotectants on the cryopreservation of agouti (Dasyprocta leporina) epididymal sperm. We used 16 pairs of testes-epididymis complexes of sexually mature animals. We immediately evaluated epididymal sperm obtained by retrograde flushing for concentration, motility, vigor, viability, osmotic response, and morphology. Samples were extended in a coconut water extender plus 20% egg yolk, containing glycerol, ethylene glycol, dimethylsulfoxide - DMSO, or dimethylformamide. Finally, samples were stored in 0.25 mL straws, frozen in liquid nitrogen, and thawed after one week, being reevaluated and assessed for membrane integrity using fluorescent probes. The higher values for postthawing sperm motility, vigor, and membrane integrity were achieved by the usage of glycerol, when compared to ethylene glycol and dimethylformamide (P < 0.05); however, no differences were found between glycerol and DMSO (P > 0.05). All cryoprotectants provided a similar effect on the preservation of sperm morphology, osmotic response, and viability (P > 0.05). Therefore, here onwards, there was testing of glycerol and DMSO at 3 and 6% concentrations using the same freezing-thawing protocol reported previously. As the main result, DMSO at 6% concentration provided a decrease in sperm parameters, as well as in the chromatin integrity and in the binding capability of sperm. In conclusion, glycerol 3 or 6% and DMSO 3% can be used as alternative cryoprotectants for agouti epididymal sperm cryopreservation.

  19. Sperm nuclear expansion and meiotic maturation in normal and gynogenetic eggs of the scallop, Chlamys farreri

    NASA Astrophysics Data System (ADS)

    Pan, Ying; Li, Qi; Yu, Ruihai; Wang, Rucai

    2008-02-01

    Sperm nuclear expansion, meiosis and the association of the male and female pronuclei leading to the four-cell stage in normal Chlamys farreri eggs were observed under a fluorescence microscope. The effects of ultraviolet (UV) irradiation on the fertilizing sperm were also examined. Both normal and UV-irradiated sperm nuclei enlarged at three distinct phases (phase A, metaphase I; phase B, polar body formation; and phase C, female pronuclear development and expansion) that were temporally correlated with meiotic process of the maternal chromosomes. Sperm nuclei underwent a rapid, initial enlargement during phase A, but condensed slightly during phase B, then re-enlarged during phase C. The effects of UV irradiation were not apparent during transformation of the sperm nucleus into a male pronucleus, and there was not any apparent effect on meiotic maturation and development of the female pronucleus. However, the rate of expansion of the UV-irradiated sperm nuclei and the size of male pronuclei were reduced apparently. Unlike the female pronucleus, the male pronucleus derived from sperm genome inactivated by UV irradiation did not form chromosomes, but became a dense chromatin body (DCB). At mitotic anaphase, DCB did not participate in the karyokinesis of the first cleavage as evidenced by chromosomal nondisjunction, demonstrating the effectiveness of using UV irradiation to induce gynogenetic scallop embryos.

  20. Application of x-rays to the analysis of DNA packaging in mammalian sperm.

    SciTech Connect

    Balhorn, R.; Corzett, M.; Allen, M.; Lee, C.; Barbee, T.; Koch, J.; MacGowan, B.; Matthews, D.; Mrowka, S.; Trebes, J.; Experimental Facilities Division; LLNL; Univ. of California, Berkeley; Univ. of California, San Francisco; LBNL Lab.

    1993-01-01

    X-ray microscopy has been performed on unlabeled and gold labeled rat sperm nuclei using a tantalum x-ray laser (44.83 angstrom) and an x-ray zone plate lens. Transmission images of nuclei labeled with gold using an antibody to protamine 1 show large clusters of gold as well as individual 400 angstrom gold particles coating the surface of the chromatin. Images of the same nuclei obtained with a scanning x-ray microscope demonstrate that the initial exposure of the sperm nucleus to a photon intensity of 3.0 X 10{sup 14} W/cm{sup 2} for a duration of 500 ps did not destroy or grossly distort their structure. Other nuclei labeled with an antibody to protamine 2 were partially decondensed during the labeling process and contained large vacuole-like regions. Images were also obtained of unlabeled sperm nuclei. Data obtained from one unlabeled nucleus imaged with both the atomic force and x-ray laser microscopes was used to determine the thickness of the chromatin and estimate the amount of water that must be associated with the DNA-protamine complex in air-dried nuclei. These results suggest that even air-dried sperm nuclei are extensively hydrated and that tightly bound water may comprise as much as one-third of the volume of the dried sperm nucleus.

  1. Responses of testis, epididymis, and sperm of pubertal rats exposed to functionalized multiwalled carbon nanotubes.

    PubMed

    Farombi, Ebenezer O; Adedara, Isaac A; Forcados, Gilead E; Anao, Osemudiamen O; Agbowo, Agatha; Patlolla, Anita K

    2016-05-01

    The present study investigated the response of testes, epididymides and sperm in pubertal Wistar rats following exposure to 0, 0.25, 0.5, 0.75, and 1.0 mg kg(-1) functionalized multi-walled carbon nanotubes (f-MWCNTs) for 5 days. The results showed that administration of (f-MWCNTs) significantly increased the activities of superoxide dismutase, catalase, and glutathione peroxidase in a dose-dependent manner in both testes and sperm compared with control group. Moreover, the significant decrease in the activity of glutathione-S-transferase and glutathione level was accompanied with significant elevation in the levels of hydrogen peroxide and malondialdehyde in both testes and sperm of (f-MWCNTs)-treated rats. The spermiogram of (f-MWCNTs)-treated rats indicated significant decrease in epididymal sperm number, sperm progressive motility, testicular sperm number and daily sperm production with elevated sperm abnormalities when compared with the control. Exposure to (f-MWCNTs) decreased plasma testosterone level and produced marked morphological changes including decreased geminal epithelium, edema, congestion, reduced spermatogenic cells and focal areas of tubular degeneration in the testes. The lumen of the epididymides contained reduced sperm cells and there was mild to severe hyperplasia epithelial cells lining the duct of the epididymis. Collectively, pubertal exposure of male rats to (f-MWCNTs) elicited oxidative stress response resulting in marked testicular and epididymides dysfunction.

  2. Sperm of patients with severe asthenozoospermia show biochemical, molecular and genomic alterations.

    PubMed

    Bonanno, Oriana; Romeo, Giulietta; Asero, Paola; Pezzino, Franca Maria; Castiglione, Roberto; Burrello, Nunziatina; Sidoti, Giuseppe; Frajese, Giovanni Vanni; Vicari, Enzo; D'Agata, Rosario

    2016-12-01

    The multifactorial pathological condition, that is, severe low sperm motility is a frequent cause of infertility. However, mechanisms underlying the development of this condition are not completely understood. Single abnormalities have been reported in sperm of patients with asthenozoospermia. In this study, we characterized, in 22 normozoospermic men and in 37 patients with asthenozoospermia, biochemical, molecular and genomic abnormalities that frequently occur in sperm of patients with asthenozoospermia. We evaluated a panel of sperm biomarkers that may affect the motility and fertilizing ability of sperm of patients with severe asthenozoospermia. Since reactive oxygen species (ROS) production is involved in the pathogenesis of such sperm abnormalities, we determined the association between ROS production and sperm abnormalities. High percentage of patients with severe asthenozoospermia showed increased basal and stimulated ROS production. Moreover, these patients showed increased mitochondrial DNA (mtDNA) copy number but decreased mtDNA integrity and they were associated with elevated ROS levels. Furthermore, mitochondrial membrane potential was also significantly decreased and again associated with high ROS production in these patients. However, the rate of nuclear DNA fragmentation was increased only in less than one-fifth of these patients. An important cohort of these patients showed multiple identical biochemical, molecular and genomic abnormalities, which are typical manifestations of oxidative stress. The most frequent association was found in patients with high ROS levels, increased mtDNA copy number and decreased integrity, and low MMP. A smaller cohort of the aforementioned patients also showed nDNA fragmentation. Therefore, patients with asthezoospermia likely present reduced fertilizing potential because of such composed abnormalities.

  3. The Effect of Oxidative Stress on Thawed Bulk-Sorted Red Deer Sperm.

    PubMed

    Anel-López, L; García-Álvarez, O; Parrilla, I; Del Olmo, D; Fernández-Santos, M R; Soler, A J; Maroto-Morales, A; Ortiz, J A; Alkmin, D V; Tarantini, T; Roca, J; Martínez, E A; Vazquez, J M; Garde, J J

    2016-06-01

    The aims of this study were to assess the effects of the sex-sorting process on post-thaw sperm quality as well as on induced oxidative stress damage (H2 O2 0 mm = H000; H2 O2 50 mm = H050; H2 O2 100 mm = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non-sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H2 O2 increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm-sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non-sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.

  4. Comparative evidence for the evolution of sperm swimming speed by sperm competition and female sperm storage duration in passerine birds.

    PubMed

    Kleven, Oddmund; Fossøy, Frode; Laskemoen, Terje; Robertson, Raleigh J; Rudolfsen, Geir; Lifjeld, Jan T

    2009-09-01

    Sperm swimming speed is an important determinant of male fertility and sperm competitiveness. Despite its fundamental biological importance, the underlying evolutionary processes affecting this male reproductive trait are poorly understood. Using a comparative approach in a phylogenetic framework, we tested the predictions that sperm swim faster with (1) increased risk of sperm competition, (2) shorter duration of female sperm storage, and (3) increased sperm length. We recorded sperm swimming speed in 42 North American and European free-living passerine bird species, representing 35 genera and 16 families. We found that sperm swimming speed was positively related to the frequency of extrapair paternity (a proxy for the risk of sperm competition) and negatively associated with clutch size (a proxy for the duration of female sperm storage). Sperm swimming speed was unrelated to sperm length, although sperm length also increased with the frequency of extrapair paternity. These results suggest that sperm swimming speed and sperm length are not closely associated traits and evolve independently in response to sperm competition in passerine birds. Our findings emphasize the significance of both sperm competition and female sperm storage duration as evolutionary forces driving sperm swimming speed.

  5. Spermiogenesis and chromatin condensation in the common tree shrew, Tupaia glis.

    PubMed

    Suphamungmee, Worawit; Wanichanon, Chaitip; Vanichviriyakit, Rapeepun; Sobhon, Prasert

    2008-03-01

    We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80-100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15-16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12-16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells.

  6. Mice produced by mitotic reprogramming of sperm injected into haploid parthenogenotes

    PubMed Central

    Suzuki, Toru; Asami, Maki; Hoffmann, Martin; Lu, Xin; Gužvić, Miodrag; Klein, Christoph A.; Perry, Anthony C. F.

    2016-01-01

    Sperm are highly differentiated and the activities that reprogram them for embryonic development during fertilization have historically been considered unique to the oocyte. We here challenge this view and demonstrate that mouse embryos in the mitotic cell cycle can also directly reprogram sperm for full-term development. Developmentally incompetent haploid embryos (parthenogenotes) injected with sperm developed to produce healthy offspring at up to 24% of control rates, depending when in the embryonic cell cycle injection took place. This implies that most of the first embryonic cell cycle can be bypassed in sperm genome reprogramming for full development. Remodelling of histones and genomic 5′-methylcytosine and 5′-hydroxymethylcytosine following embryo injection were distinct from remodelling in fertilization and the resulting 2-cell embryos consistently possessed abnormal transcriptomes. These studies demonstrate plasticity in the reprogramming of terminally differentiated sperm nuclei and suggest that different epigenetic pathways or kinetics can establish totipotency. PMID:27623537

  7. Effects of oral exposure to silver nanoparticles on the sperm of rats.

    PubMed

    Lafuente, D; Garcia, T; Blanco, J; Sánchez, D J; Sirvent, J J; Domingo, J L; Gómez, M

    2016-04-01

    It has been demonstrated that exposure to silver nanoparticles (AgNPs) can induce toxicological effects in rodents. In this study, we investigated whether sub-chronic oral exposure to different doses of polyvinil pyrrolidone (PVP)-coated AgNPs (PVP-AgNPs) (50, 100 and 200mg/kg/day) could induce harmful effects on epididymal sperm rat parameters. Sperm motility, viability and morphology were examined. Moreover, a histological evaluation of testis and epididymis was also performed. High doses of PVP-AgNPs showed higher sperm morphology abnormalities, while a progressive, but not significant effect, was observed in other sperm parameters. The current results suggest that oral sub-chronic exposure to PVP-AgNPs induces slight toxicological effects in sperm rat parameters.

  8. Liquid storage of equine semen: Assessing the effect of d-penicillamine on longevity of ejaculated and epididymal stallion sperm.

    PubMed

    Brogan, P T; Beitsma, M; Henning, H; Gadella, B M; Stout, T A E

    2015-08-01

    Short-term storage of equine sperm at 5°C in an extender containing milk and/or egg yolk components is common practice in the equine breeding industry. Sperm motility, viability, DNA integrity and, consequently, fertilizing ability decline over time, partly due to reactive oxygen species (ROS) generation. We investigated whether adding the anti-oxidant d-penicillamine to a commercial milk/egg yolk extender delayed the decrease in semen quality. Semen was recovered on four consecutive days from eight 3-year old Warmblood stallions. On day 5, seven of the stallions were castrated and sperm recovered from the caudae epididymides. Ejaculated samples were split, and one portion was centrifuged and re-suspended to reduce seminal plasma content. All samples were diluted to 50millionsperm/ml and divided into two portions, one of which was supplemented with 0.5mM d-penicillamine. After 48h, 96h, 144h and 192h storage, sperm motility was assessed by computer-assisted semen analysis (CASA), viability by SYBR14/PI staining, and DNA integrity using the sperm chromatin structure assay (SCSA). d-Penicillamine had no effect on motility of ejaculated sperm (P>0.05) but reduced total and progressive motility of epididymal sperm. Sperm chromatin integrity was not influenced by storage time, seminal plasma or d-penicillamine. In short, adding d-penicillamine to a commercial semen extender was neither beneficial nor detrimental to the maintenance of quality in ejaculated semen stored at 5°C. The negative effect on motility of epididymal sperm may reflect differences in (membrane) physiology of spermatozoa that have not been exposed to seminal plasma.

  9. Turbulence of swarming sperm

    NASA Astrophysics Data System (ADS)

    Creppy, Adama; Praud, Olivier; Druart, Xavier; Kohnke, Philippa L.; Plouraboué, Franck

    2015-09-01

    Collective motion of self-sustained swarming flows has recently provided examples of small-scale turbulence arising where viscous effects are dominant. We report the first observation of universal enstrophy cascade in concentrated swarming sperm consistent with a body of evidence built from various independent measurements. We found a well-defined k-3 power-law decay of a velocity field power spectrum and relative dispersion of small beads consistent with theoretical predictions in 2D turbulence. Concentrated living sperm displays long-range, correlated whirlpool structures of a size that provides an integral scale of turbulence. We propose a consistent explanation for this quasi-2D turbulence based on self-structured laminated flow forced by steric interactions and alignment, a state of active matter that we call "swarming liquid crystal." We develop scaling arguments consistent with this interpretation.

  10. Sperm, Clinics, and Parenthood

    PubMed Central

    2016-01-01

    Abstract In this article I examine a recent approach to regulating assisted reproduction, whereby use of some kind of medical intervention ‘triggers’ laws governing legal parenthood that are more favourable to intending parents and sperm providers. I argue that although perhaps an improvement on the previous legal framework, these laws are problematic for three important reasons. First, they are prone to violating parental rights and unjustly imposing substantial burdens on individuals. Second, they are discriminatory. Third, even if we take a pragmatic approach to the question of parenthood in these cases, these laws fail to properly consider the welfare interests of children. Finally, I conclude by showing that my argument does not entail adopting a laissez‐fair attitude to conception using third‐party sperm. PMID:27523389

  11. Decreased activity of superoxide dismutase in the seminal plasma of infertile men correlates with increased sperm deoxyribonucleic acid fragmentation during the first hours after sperm donation.

    PubMed

    Wdowiak, Artur; Bakalczuk, Szymon; Bakalczuk, Grzegorz

    2015-07-01

    Sperm DNA fragmentation varies between individuals and is more pronounced with increased patient age and time after sperm donation. The intensification of DNA fragmentation depends on the balance of the oxidoreductive system, which is regulated mainly by two enzymes - superoxide dismutase (SOD) and catalase. The objective of this study was to determine the relationship between sperm DNA fragmentation dynamics, fertility and seminal SOD and catalase activity. The study was conducted in 2013 and 2014 at the Non-Public Health Care Unit 'Ovum Reproduction and Andrology' in Lublin, Lublin, Poland, and covered 218 men aged 25-35 (85 fertile and 133 patients treated for infertility). Percentage of fragmented DNA was measured in a modified chromatin dispersion test at four time points after sperm donation (t = 0, 3, 6, 12 h). SOD and catalase activities were determined spectrophotometrically. We confirmed that the activity of SOD in the seminal plasma of men with reproductive disorders was lower compared with fertile men. Conversely, no significant correlations were found between fertility and catalase activity. Sperm DNA of infertile males was initially more fragmented than fertile male sperm DNA. SOD and catalase activity did not correlate with the degree of DNA fragmentation in fertile men. In men with reproductive disorders, the rate of DNA fragmentation was slow within first 3 h after sperm donation and then increased between 6 and 12 h. In this group of infertile men, those with higher SOD activity had a lower DNA fragmentation index (DFI) after 12 h, and a reduced rate of intensity of fragmentation from 6 to 12 h. Alternatively, higher catalase activity among men treated for infertility was accompanied by higher initial DFI and higher rate of DNA fragmentation from 6 to 12 h. These results highlight the importance of determining a proper time window between sperm donation and procedures of assisted reproductive technology.

  12. [Status quo of the researches on the biological effect of electromagnetic radiation on the testis and epididymal sperm].

    PubMed

    Gao, Xiao-fang; Wang, Shui-ming; Peng, Rui-yun

    2007-09-01

    The testis is highly sensitive to electromagnetic radiation. Sperm is the passer of male genetic material and electromagnetic radiation may cause structural and functional injury to the testis, including motility reduction, abnormality increase and ultrastructural alteration of epididymal sperm. Energy metabolism disorder in spermatogenic cells, enhancement of lipid peroxidation in the testis, excessive expression of inflammatory factors and abnormality of genetic transcription may be responsible for injury to the testis and epididymal sperm. This paper reviews the progress made in this field and the preventive measures against the injury.

  13. Sperm function test

    PubMed Central

    Talwar, Pankaj; Hayatnagarkar, Suryakant

    2015-01-01

    With absolute normal semen analysis parameters it may not be necessary to shift to specialized tests early but in cases with borderline parameters or with history of fertilization failure in past it becomes necessary to do a battery of tests to evaluate different parameters of spermatozoa. Various sperm function tests are proposed and endorsed by different researchers in addition to the routine evaluation of fertility. These tests detect function of a certain part of spermatozoon and give insight on the events in fertilization of the oocyte. The sperms need to get nutrition from the seminal plasma in the form of fructose and citrate (this can be assessed by fructose qualitative and quantitative estimation, citrate estimation). They should be protected from the bad effects of pus cells and reactive oxygen species (ROS) (leukocyte detection test, ROS estimation). Their number should be in sufficient in terms of (count), structure normal to be able to fertilize eggs (semen morphology). Sperms should have intact and functioning membrane to survive harsh environment of vagina and uterine fluids (vitality and hypo-osmotic swelling test), should have good mitochondrial function to be able to provide energy (mitochondrial activity index test). They should also have satisfactory acrosome function to be able to burrow a hole in zona pellucida (acrosome intactness test, zona penetration test). Finally, they should have properly packed DNA in the nucleus to be able to transfer the male genes (nuclear chromatic decondensation test) to the oocyte during fertilization. PMID:26157295

  14. Cytometry of mammalian sperm

    SciTech Connect

    Gledhill, B.L.

    1983-10-11

    Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. The accessibility of male cells makes them well suited for analytical cytology. We might automate the process of determining sperm morphology but should not do so solely for increased speed. Rather, richer tangible benefits will derive from cytometric evaluation through increased sensitivity, reduced subjectivity, standardization between investigators and laboratories, enhanced archival systems, and the benefits of easily exchanged standardized data. Inroads on the standardization of assays for motility and functional integrity are being made. Flow cytometric analysis of total DNA content of individual sperm is an insensitive means to detect exposure to reproductive toxins because of the small size and low frequency of the DNA content errors. Flow cytometry can be applied to determine the proportions of X- and Y-sperm in semen samples.

  15. Influence of M-phase chromatin on the anisotropy of microtubule asters

    PubMed Central

    1996-01-01

    In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long- range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long- range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length. PMID:8601601

  16. Rapid freezing without cooling equilibration in canine sperm.

    PubMed

    Kim, Suhee; Lee, Yongcheol; Yang, Honghyun; Kim, Yong-Jun

    2012-01-01

    The aim of this study was to develop a rapid method of canine semen freezing without cooling equilibration using treatment with different cryoprotectant agents (CPAs) and freezing in liquid nitrogen (LN(2)) vapor in a 0.5-mL straw via modifying vitrification. Ejaculates from eight beagle dogs were frozen with different CPAs (CPA-free, 5% glycerol, 5% ethylene glycol, and 10% ethylene glycol) and freezing times (direct plunging into LN(2) or freezing for 1, 2, 3, or 10 min in LN(2) vapor before plunging into LN(2)). Frozen-thawed sperm were evaluated for motility, viability, normal morphology, and plasma- and acrosome-membrane integrities. The 5% glycerol treatment resulted in improved sperm motility, plasma-membrane integrity and acrosome-membrane integrity (P<0.05). Freezing in LN(2) vapor showed improved sperm motility, viability, and plasma membrane integrity (P<0.05), and freezing for more than 2 min in LN(2) vapor increased acrosome-membrane integrity compared with direct plunging into LN(2) (P<0.05). The direct plunging into LN(2) showed no motile sperm. However, freezing for more than 2 min in LN(2) vapor increased the total abnormalities compared to direct plunging into LN(2) (P<0.05). In conclusion, use of 5% glycerol and freezing in LN(2) vapor were essential for the rapid freezing of canine sperm without cooling equilibration. In particular, holding for 2 min in LN(2) vapor was sufficient to yield successful rapid freezing. This rapid freezing method is simple and effective in canine sperm and would be helpful to offer information for trial of vitrification in large volumes of canine sperm.

  17. Neutron scatter studies of chromatin structures related to functions. Technical progress report, November 1, 1991--May 15, 1992

    SciTech Connect

    Bradbury, E.M.

    1992-06-01

    We have made considerable progress in chromatin reconstitution with very lysine rich histone H1/H5 and in understanding the dynamics of nucleosomes. A ferromagnetic fluid was developed to align biological molecules for structural studies using small-angle-neutron-scattering. We have also identified and characterized in intrinsically bent DNA region flaking the RNA polymerase I binding site of the ribosomal RNA gene in Physarum Polycephalum. Finally projects in progress are in the areas of studying the interactions of histone H4 amino-terminus peptide 1-23 and acetylated 1-23 peptide with DNA using thermal denaturation; study of GGAAT repeats found in human centromeres using high resolution Nuclear Magnetic Resonance and nuclease sentivity assay; and the role of histones and other sperm specific proteins with sperm chromatin.

  18. Neutron scatter studies of chromatin structures related to functions. Technical progress report, November 1, 1991--May 15, 1992

    SciTech Connect

    Bradbury, E.M.

    1992-11-01

    Despite of setbacks in the lack of neutrons for the proposed We have made considerable progress in chromatin reconstitution with the VLR histone H1/H5 and in understanding the dynamics of nucleosomes. A ferromagnetic fluid was developed to align biological molecules for structural studies using small-angle-neutron-scattering. We have also identified and characterized an intrinsically bent DNA region flanking the RNA polymerase I binding site of the ribosomal RNA gene in Physarum Polycephalum. Finally projects in progress are in the areas of studying the interatctions of histone H4 amino-terminus peptide 1-23 and acetylated 1-23 peptide with DNA using thermal denaturation; study of GGAAT repeats found in human centromeres using high resolution Nuclear magnetic Resonance and nuclease sentivity assay; and the role of histones and other sperm specific proteins with sperm chromatin.

  19. ETOPOSIDE INDUCES CHROMOSOMAL ABNORMALITIES IN SPERMATOCYTES AND SPERMATOGONIAL STEM CELLS

    SciTech Connect

    Marchetti, F; Pearson, F S; Bishop, J B; Wyrobek, A J

    2005-07-15

    Etoposide (ET) is a chemotherapeutic agent widely used in the treatment of leukemia, lymphomas and many solid tumors, such as testicular and ovarian cancers, that affect patients in their reproductive years. The purpose of the study was to use sperm FISH analyses to characterize the long-term effects of ET on male germ cells. We used a mouse model to characterize the induction of chromosomal aberrations (partial duplications and deletions) and whole chromosomal aneuploidies in sperm of mice treated with a clinical dose of ET. Semen samples were collected at 25 and 49 days after dosing to investigate the effects of ET on meiotic pachytene cells and spermatogonial stem-cells, respectively. ET treatment resulted in major increases in the frequencies of sperm carrying chromosomal aberrations in both meiotic pachytene (27- to 578-fold) and spermatogonial stem-cells (8- to 16-fold), but aneuploid sperm were induced only after treatment of meiotic cells (27-fold) with no persistent effects in stem cells. These results demonstrate that male meiotic germ cells are considerably more sensitive to ET than spermatogonial stem-cell and that increased frequencies of sperm with structural aberrations persist after spermatogonial stem-cell treatment. These findings predict that patients who undergo chemotherapy with ET may have transient elevations in the frequencies of aneuploid sperm, but more importantly, may have persistent elevations in the frequencies of sperm with chromosomal aberrations, placing them at higher risk for abnormal reproductive outcomes long after the end of their chemotherapy.

  20. Quantitative analysis of radiation-induced changes in sperm morphology

    SciTech Connect

    Young, I.T.; Gledhill, B.L.; Lake, S.; Wyrobek, A.J.

    1982-09-01

    When developing spermatogenic cells are exposed to radiation, chemical carcinogens or mutagens, the transformation in the morphology of the mature sperm can be used to determine the severity of the exposure. In this study five groups of mice with three mice per group received testicular doses of X irradiation at dosage levels ranging from 0 rad to 120 rad. A random sample of 100 mature sperm per mouse was analyzed five weeks later for the quantitative morphologic transformation as a function of dosage level. The cells were stained with gallocyanin chrome alum (GCA) so that only the DNA in the sperm head was visible. The ACUity quantitative microscopy system at Lawrence Livermore National Laboratory was used to scan the sperm at a sampling density of 16 points per linear micrometer and with 256 brightness levels per point. The contour of each cell was extracted using conventional thresholding techniques on the high-contrast images. For each contour a variety of shape features was then computed to characterize the morphology of that cell. Using the control group and the distribution of their shape features to establish the variability of a normal sperm population, the 95% limits on normal morphology were established. Using only four shape features, a doubling dose of approximately 39 rad was determined. That is, at 39 rad exposure the percentage of abnormal cells was twice that occurring in the control population. This compared to a doubling dose of approximately 70 rad obtained from a concurrent visual procedure.

  1. The influence of direct mobile phone radiation on sperm quality

    PubMed Central

    Gorpinchenko, Igor; Nikitin, Oleg; Shulyak, Alexander

    2014-01-01

    Introduction It is impossible to imagine a modern socially–active man who does not use mobile devices and/or computers with Wi–Fi function. The effect of mobile phone radiation on male fertility is the subject of recent interest and investigations. The aim of this study was to investigate the direct in vitro influence of mobile phone radiation on sperm DNA fragmentation and motility parameters in healthy subjects with normozoospermia. Material and methods 32 healthy men with normal semen parameters were selected for the study. Each sperm sample was divided into two equal portions (A and B). Portions A of all involved men were placed for 5 hours in a thermostat, and portions B were placed into a second thermostat for the same period of time, where a mobile phone in standby/talk mode was placed. After 5 hours of incubation the sperm samples from both thermostats were re–evaluated regarding basic motility parameters. The presence of DNA fragmentation in both A and B portions of each sample was determined each hour using a standard sperm chromatin dispersion test. Results The number of spermatozoa with progressive movement in the group, influenced by electromagnetic radiation, is statistically lower than the number of spermatozoa with progressive movement in the group under no effect of the mobile phone. The number of non–progressive movement spermatozoa was significantly higher in the group, which was influenced by cell phone radiation. The DNA fragmentation was also significantly higher in this group. Conclusions A correlation exists between mobile phone radiation exposure, DNA–fragmentation level and decreased sperm motility. PMID:24982785

  2. Aneuploidy in sperm of Hodgkin`s disease patients receiving NOVP chemotherapy

    SciTech Connect

    Robbins, W.A.; Cassel, M.J.; Wyrobek, A.J.

    1994-09-01

    Induction of genetic damage in germ cells of young patients receiving chemo- or radiotherapy for cancers with probable cure, such as Hodgkin`s disease, is cause for concern. These young patients may someday desire children, and germ cell alterations presenting as numerical chromosomal abnormalities in sperm may place their future offspring at risk. To address this concern, we measured aneuploidy in sperm from eight young Hodgkin`s disease patients: four pre-treatment, four during treatment, and three over a 45 month period following treatment with NOVP (Novantrone, Oncovin, Vinblastine and Prednisone). Patients ranged in stage of disease from IA-IIEB and none had received prior radiation or chemotherapy. Using multi-chromosome sperm FISH with repetitive sequence probes specific for chromosomes X, Y and 8, we found a significant 2-4 fold increase in particular numerical chromosomal abnormalities during treatment which were limited in persistence post-treatment. Additionally, pre-treatment Hodgkin`s disease patients showed elevations in some numerical chromosomal abnormalities when compared to a healthy reference group. In several men, the fraction of aneuploid sperm did not return to healthy reference group levels even after completion of therapy. These results show that elevated sperm aneuploidy occurs in germ cells of young cancer patients during chemotherapy and suggest caution to prevent conceptions during this period. The elevated sperm aneuploidy appears transient, but in some cases never returns to healthy reference group levels.

  3. Histone lysine methylation and chromatin replication.

    PubMed

    Rivera, Carlos; Gurard-Levin, Zachary A; Almouzni, Geneviève; Loyola, Alejandra

    2014-12-01

    In eukaryotic organisms, the replication of the DNA sequence and its organization into chromatin are critical to maintain genome integrity. Chromatin components, such as histone variants and histone post-translational modifications, along with the higher-order chromatin structure, impact several DNA metabolic processes, including replication, transcription, and repair. In this review we focus on lysine methylation and the relationships between this histone mark and chromatin replication. We first describe studies implicating lysine methylation in regulating early steps in the replication process. We then discuss chromatin reassembly following replication fork passage, where the incorporation of a combination of newly synthesized histones and parental histones can impact the inheritance of lysine methylation marks on the daughter strands. Finally, we elaborate on how the inheritance of lysine methylation can impact maintenance of the chromatin landscape, using heterochromatin as a model chromatin domain, and we discuss the potential mechanisms involved in this process.

  4. Chromatin Structure in Telomere Dynamics

    PubMed Central

    Galati, Alessandra; Micheli, Emanuela; Cacchione, Stefano

    2013-01-01

    The establishment of a specific nucleoprotein structure, the telomere, is required to ensure the protection of chromosome ends from being recognized as DNA damage sites. Telomere shortening below a critical length triggers a DNA damage response that leads to replicative senescence. In normal human somatic cells, characterized by telomere shortening with each cell division, telomere uncapping is a regulated process associated with cell turnover. Nevertheless, telomere dysfunction has also been associated with genomic instability, cell transformation, and cancer. Despite the essential role telomeres play in chromosome protection and in tumorigenesis, our knowledge of the chromatin structure involved in telomere maintenance is still limited. Here we review the recent findings on chromatin modifications associated with the dynamic changes of telomeres from protected to deprotected state and their role in telomere functions. PMID:23471416

  5. Easy sperm processing technique allowing exclusive accumulation and later usage of DNA-strandbreak-free spermatozoa.

    PubMed

    Ebner, T; Shebl, O; Moser, M; Mayer, R B; Arzt, W; Tews, G

    2011-01-01

    Sperm DNA fragmentation is increased in poor-quality semen samples and correlates with failed fertilization, impaired preimplantation development and reduced pregnancy outcome. Common sperm preparation techniques may reduce the percentage of strandbreak-positive spermatozoa, but, to date, there is no reliable approach to exclusively accumulate strandbreak-free spermatozoa. To analyse the efficiency of special sperm selection chambers (Zech-selectors made of glass or polyethylene) in terms of strandbreak reduction, 39 subfertile men were recruited and three probes (native, density gradient and Zech-selector) were used to check for strand breaks using the sperm chromatin dispersion test. The mean percentage of affected spermatozoa in the ejaculate was 15.8 ± 7.8% (range 5.0–42.1%). Density gradient did not significantly improve the quality of spermatozoa selected(14.2 ± 7.0%). However, glass chambers completely removed 90% spermatozoa showing strand breaks and polyethylene chambers removed 76%. Both types of Zech-selectors were equivalent in their efficiency, significantly reduced DNA damage (P < 0.001) and,with respect to this, performed better than density gradient centrifugation (P < 0.001). As far as is known, this is the first report ona sperm preparation technique concentrating spermatozoa unaffected in terms of DNA damage. The special chambers most probably select for sperm motility and/or maturity.

  6. Exposure to bisphenol A (BPA) in Wistar rats reduces sperm quality with disruption of ERK signal pathway.

    PubMed

    Li, Juan; Mao, Rui; Zhou, Qin; Ding, Ling; Tao, Jin; Ran, Mao-Mei; Gao, Er-Sheng; Yuan, Wei; Wang, Jin-Tao; Hou, Li-Fang

    2016-01-01

    Bisphenol A (BPA) is an estrogenic environmental toxin widely used in the production of plastics and ubiquitous human exposure to this chemical has been proposed to be a potential risk to human health. Exposure to BPA can negatively impact sperm quality. However, the mechanism remains largely unknown. The objectives of this study were to assess the role of BPA on sperm quality and explore the possible mechanisms. The Wistar male rats (aged 28 days) were administered BPA by oral gavage for 28 days at dose of 50, 100 and 200 mg/kg/day; meanwhile, the negative control with corn oil (0 mg/kg/day BPA) and positive control with E2 at the dose of 100 μg/kg/day. The sperm density, sperm activity and sperm survival rate were analyzed byCASA system, and the sperm abnormality rate was analyzed by improved Papanicolaou stained. The protein expression levels of Src/p-Src, ERK1/2, p-ERK1/2 and CREB/p-CREB were detected by Western bolt. The results showed that the body weight gain, testes weight, testis coefficient, sperm density, sperm activity, sperm survival rate and protein expression levels of p-ERK1, p-ERK2 and p-CREB decreased, but the sperm abnormality rate increased with increasing BPA concentrations. There were positive correlations between sperm density, sperm activity and sperm survival rate with protein expression levels of p-ERK1, p-ERK2 and p-CREB, and negative correlations between sperm abnormality rate with the protein expression levels of p-ERK1, p-ERK2 and p-CREB. Results from the structural equation model demonstrated that BPA retained a significant negative effect to p-ERK, whereas p-ERK retained a significant positive effect to sperm quality and acted as the mediate variable. This study provides a novel insight regarding the potential role of p-ERK1 and p-ERK2 protein kinase on reproductive toxicity of BPA. The adverse effects of BPA on adult male sperm quality may be through the induction of the disruption of ERK signal pathway. However, additional

  7. Identification of genomic features in environmentally induced epigenetic transgenerational inherited sperm epimutations.

    PubMed

    Guerrero-Bosagna, Carlos; Weeks, Shelby; Skinner, Michael K

    2014-01-01

    A variety of environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. The process involves exposure of a gestating female and the developing fetus to environmental factors that promote permanent alterations in the epigenetic programming of the germline. The molecular aspects of the phenomenon involve epigenetic modifications (epimutations) in the germline (e.g. sperm) that are transmitted to subsequent generations. The current study integrates previously described experimental epigenomic transgenerational data and web-based bioinformatic analyses to identify genomic features associated with these transgenerationally transmitted epimutations. A previously identified genomic feature associated with these epimutations is a low CpG density (<12/100bp). The current observations suggest the transgenerational differential DNA methylation regions (DMR) in sperm contain unique consensus DNA sequence motifs, zinc finger motifs and G-quadruplex sequences. Interaction of molecular factors with these sequences could alter chromatin structure and accessibility of proteins with DNA methyltransferases to alter de novo DNA methylation patterns. G-quadruplex regions can promote the opening of the chromatin that may influence the action of DNA methyltransferases, or factors interacting with them, for the establishment of epigenetic marks. Zinc finger binding factors can also promote this chromatin remodeling and influence the expression of non-coding RNA. The current study identified genomic features associated with sperm epimutations that may explain in part how these sites become susceptible for transgenerational programming.

  8. Choline Dehydrogenase Polymorphism rs12676 Is a Functional Variation and Is Associated with Changes in Human Sperm Cell Function

    PubMed Central

    Johnson, Amy R.; Lao, Sai; Wang, Tongwen; Galanko, Joseph A.; Zeisel, Steven H.

    2012-01-01

    Approximately 15% of couples are affected by infertility and up to half of these cases arise from male factor infertility. Unidentified genetic aberrations such as chromosomal deletions, translocations and single nucleotide polymorphisms (SNPs) may be the underlying cause of many cases of idiopathic male infertility. Deletion of the choline dehydrogenase (Chdh) gene in mice results in decreased male fertility due to diminished sperm motility; sperm from Chdh−/− males have decreased ATP concentrations likely stemming from abnormal sperm mitochondrial morphology and function in these cells. Several SNPs have been identified in the human CHDH gene that may result in altered CHDH enzymatic activity. rs12676 (G233T), a non-synonymous SNP located in the CHDH coding region, is associated with increased susceptibility to dietary choline deficiency and risk of breast cancer. We now report evidence that this SNP is also associated with altered sperm motility patterns and dysmorphic mitochondrial structure in sperm. Sperm produced by men who are GT or TT for rs12676 have 40% and 73% lower ATP concentrations, respectively, in their sperm. rs12676 is associated with decreased CHDH protein in sperm and hepatocytes. A second SNP located in the coding region of IL17BR, rs1025689, is linked to altered sperm motility characteristics and changes in choline metabolite concentrations in sperm. PMID:22558321

  9. New method to estimate the possibility of natural pregnancy using computer-assisted sperm analysis.

    PubMed

    Isobe, Tetsuya

    2012-12-01

    The World Health Organization (WHO) criteria, which include percent motility and sperm concentration, are the only criteria for evaluating sperm quality and conception ability. However, these criteria are insufficient to evaluate the possibility of natural pregnancy. Thus, an index that can directly evaluate the possibility of a natural pregnancy is necessary. A new sperm energy theory without approximation was developed to assess the possibility of natural pregnancy based on mechanical sperm energy. Sperm motility parameters were measured using computer-assisted sperm analysis (CASA) in 129 ejaculated semen samples from 50 men in couples diagnosed with infertility, in which no abnormalities were found in women (sterile group), and 157 ejaculated semen samples from 57 men who had already fathered children in natural pregnancies (control group). A total of 129 subjects were selected from the control group and classified as the fertile group in order of the sample measurement date. The sperm energy index (SEI) and mean sperm energy index (MEI) were accurately obtained according to the methods described by the new sperm energy theory. SEI reflects total mechanical energy of the sperm in a visual field during CASA measurements. MEI reflects the mean mechanical energy of one sperm in a measurement field. All subjects with (MEI)/(SEI) > 2 were assigned to the sterile group. The larger the SEI, the higher was the probability of predicting fertile subjects. The probability of predicting fertile subjects was approximately 60% with a SEI of > 0.5, 70% with a SEI of > 1, 80% with a SEI of > 3, and 90% with a SEI of > 6 in cases where (MEI)/(SEI) is < 2. The data support the view that this novel method can be used to estimate the possibility of a natural pregnancy.

  10. Characterization and short-term storage of Tasmanian devil sperm collected post-mortem.

    PubMed

    Keeley, T; McGreevy, P D; O'Brien, J K

    2011-09-01

    The Tasmanian devil is suffering from a severe population decline due to the fatal Devil Facial Tumour Disease (DFTD). The development of assisted reproductive technologies such as AI and long-term sperm storage could facilitate genetic management of captive insurance populations. The aim of this study was to characterise semen samples collected post-mortem, and to develop a suitable diluent for short-term preservation of devil sperm. Low numbers of sperm (1.33 ± 0.85 × 10(6) sperm per male) were extracted from the epididymides of 17 males. Devil sperm sample characteristics such as concentration and morphology were similar to other dasyurids. The most commonly observed morphological abnormalities were midpiece separation, tail curling, and tail twisting (on the axial plane). Changes in motility occurred throughout the regions of the epididymis with (mean ± SD) 29.4 ± 16.8, 46.8 ± 13.6 and 29.4 ± 18.1% of sperm exhibiting motility, and 88.9 ± 11.4, 32.0 ± 24.3 and 0.1 ± 0.2% of motile sperm exhibiting forward progressive motility in the cauda, corpus and caput, respectively. Sperm from the cauda and corpus epididymis maintained 31.7 ± 26.6 and 80.6 ± 85.9%, respectively, of initial motility after 12 h at 15 °C in a TEST yolk buffer diluent. These findings provided new information regarding devil sperm biology and short-term sperm storage; such information is necessary for future development of long-term sperm preservation methods in the Tasmanian devil.

  11. Meiotic recombination errors, the origin of sperm aneuploidy and clinical recommendations.

    PubMed

    Tempest, Helen G

    2011-02-01

    Since the early 1990s male infertility has successfully been treated by intracytoplasmic sperm injection (ICSI), nevertheless concerns have been raised regarding the genetic risk of ICSI. Chromosome aneuploidy (the presence of extra or missing chromosomes) is the leading cause of pregnancy loss and mental retardation in humans. While the majority of chromosome aneuploidies are maternal in origin, the paternal contribution to aneuploidy is clinically relevant particularly for the sex chromosomes. Given that it is difficult to study female gametes investigations are predominantly conducted in male meiotic recombination and sperm aneuploidy. Research suggests that infertile men have increased levels of sperm aneuploidy and that this is likely due to increased errors in meiotic recombination and chromosome synapsis within these individuals. It is perhaps counterintuitive but there appears to be no selection against chromosomally aneuploid sperm at fertilization. In fact the frequency of aneuploidy in sperm appears to be mirrored in conceptions. Given this information this review will cover our current understanding of errors in meiotic recombination and chromosome synapsis and how these may contribute to increased sperm aneuploidy. Frequencies of sperm aneuploidy in infertile men and individuals with constitutional karyotypic abnormalities are reviewed, and based on these findings, indications for clinical testing of sperm aneuploidy are discussed. In addition, the application of single nucleotide arrays for the analysis of meiotic recombination and identification of parental origin of aneuploidy are considered.

  12. Different Levels of DNA Methylation Detected in Human Sperms after Morphological Selection Using High Magnification Microscopy

    PubMed Central

    Cassuto, Nino Guy; Montjean, Debbie; Siffroi, Jean-Pierre; Bouret, Dominique; Marzouk, Flora; Copin, Henri; Benkhalifa, Moncef

    2016-01-01

    Objective. To analyze DNA methylation levels between two groups of spermatozoa taken from the same sample, following morphological selection by high magnification (HM) at 6100x microscopy. A prospective study was conducted and studied 876 spermatozoa from 10 randomly selected men. Sperm morphology was characterized at HM according to criteria previously established. High-scoring Score 6 and low-scoring Score 0 sperm were selected. Sperm DNA methylation level was assessed using an immunoassay method targeting 5-methylcytosine residues by fluorescence microscopy with imaging analysis system to detect DNA methylation in single spermatozoon. Results. In total, 448 S6 spermatozoa and 428 S0 spermatozoa were analyzed. A strong relationship was found between sperm DNA methylation levels and sperm morphology observed at HM. Sperm DNA methylation level in the S6 group was significantly lower compared with that in the S0 group (p < 10−6), OR = 2.4; and p < 0.001, as determined using the Wilcoxon test. Conclusion. Differences in DNA methylation levels are associated with sperm morphology variations as observed at HM, which allows spermatozoa with abnormal levels to be discarded and ultimately decrease birth defects, malformations, and epigenetic diseases that may be transmitted from sperm to offspring in ICSI. PMID:27148551

  13. Sperm proteome and reproductive technologies in mammals.

    PubMed

    Li, Chun-Jin; Wang, Dong; Zhou, Xu

    2016-10-01

    Sperm is highly differentiated cell that can be easily obtained and purified. Mature sperm is considered to be transcriptionally and translationally silent and incapable of protein synthesis. Recently, a large number of proteins have been identified in sperm from different species by using the proteomic approaches. Clinically, sperm proteins can be used as markers for male infertility due to different protein profiles identified in sperm from fertile and infertile male animals. Recent evidences have shown that the conditions of sperm preservation in vitro can also change the sperm protein profiles. This paper reviews the recent scientific publications available to address sperm proteome and their relationship with sperm cryopreservation, capacitation, fertilization, and separation of X and Y sperm. Future directions in the application of sperm proteomics to develop or optimize reproductive technologies in mammals are also discussed.

  14. Species specificity and individual variability of sea urchin sperm H2B histones.

    PubMed

    de Petrocellis, B; de Petrocellis, L; Lancieri, M; Geraci, G

    1980-01-01

    Total histones from the sperms and embryos of the sea urchins Paracentrotus lividus, Arbacia lixula, Psammechinus microtuberculatus and Sphaerechinus granularis hae been fractionated into the component molecules by electrophoretic analyses in SDS, in urea-acetic acid and in Triton-urea-acetic acid. Sperm H2B histones are in all cases different from those of the corresponding embryonic chromatins. Each sea urchin species has distinctive variants of the sperm H2B histones that are fractionated by electrophoresis in SDS acrylamide gel into two to four components forming a new class of lower mobility. This analytical method shows that individuals of the same species have different assortments of the H2B components. Electrophoretic analyses in Triton-urea also show multiple components for H2B but the patterns are similar in the different individuals.

  15. Subcellular localization of phospholipase Cζ in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation

    PubMed Central

    Escoffier, Jessica; Yassine, Sandra; Lee, Hoi Chang; Martinez, Guillaume; Delaroche, Julie; Coutton, Charles; Karaouzène, Thomas; Zouari, Raoudha; Metzler-Guillemain, Catherine; Pernet-Gallay, Karin; Hennebicq, Sylviane; Ray, Pierre F.; Fissore, Rafael; Arnoult, Christophe

    2015-01-01

    We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospholipase C, zeta (PLCζ), the sperm factor thought to induce the Ca2+ oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLCζ in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLCζ and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLCζ is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca2+ oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLCζ in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equatorial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLCζ in globozoospermic sperm is thus consistent and reinforces the role of PLCζ as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2

  16. Meiotic abnormalities in infertile males.

    PubMed

    Egozcue, J; Sarrate, Z; Codina-Pascual, M; Egozcue, S; Oliver-Bonet, M; Blanco, J; Navarro, J; Benet, J; Vidal, F

    2005-01-01

    Meiotic anomalies, as reviewed here, are synaptic chromosome abnormalities, limited to germ cells that cannot be detected through the study of the karyotype. Although the importance of synaptic errors has been underestimated for many years, their presence is related to many cases of human male infertility. Synaptic anomalies can be studied by immunostaining of synaptonemal complexes (SCs), but in this case their frequency is probably underestimated due to the phenomenon of synaptic adjustment. They can also be studied in classic meiotic preparations, which, from a clinical point of view, is still the best approach, especially if multiplex fluorescence in situ hybridization is at hand to solve difficult cases. Sperm chromosome FISH studies also provide indirect evidence of their presence. Synaptic anomalies can affect the rate of recombination of all bivalents, produce achiasmate small univalents, partially achiasmate medium-sized or large bivalents, or affect all bivalents in the cell. The frequency is variable, interindividually and intraindividually. The baseline incidence of synaptic anomalies is 6-8%, which may be increased to 17.6% in males with a severe oligozoospermia, and to 27% in normozoospermic males with one or more previous IVF failures. The clinical consequences are the production of abnormal spermatozoa that will produce a higher number of chromosomally abnormal embryos. The indications for a meiotic study in testicular biopsy are provided.

  17. Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications--a position report.

    PubMed

    Barratt, Christopher L R; Aitken, R John; Björndahl, Lars; Carrell, Douglas T; de Boer, Peter; Kvist, Ulrik; Lewis, Sheena E M; Perreault, Sally D; Perry, Melissa J; Ramos, Liliana; Robaire, Bernard; Ward, Steven; Zini, Armand

    2010-04-01

    This article reports the results of the most recent in a series of EHSRE workshops designed to synthesize the current state of the field in Andrology and provide recommendations for future work (for details see Appendix). Its focus is on methods for detecting sperm DNA damage and potential application of new knowledge about sperm chromatin organization, vulnerability and repair to improve the diagnosis and treatment of clinical infertility associated with that damage. Equally important is the use and reliability of these tests to identify the extent to which environmental contaminants or pharmaceutical agents may contribute to the incidence of sperm DNA damage and male fertility problems. A working group (for workshop details, see Appendix) under the auspices of ESHRE met in May 2009 to assess the current knowledgebase and suggest future basic and clinical research directions. This document presents a synthesis of the working group's understanding of the recent literature and collective discussions on the current state of knowledge of sperm chromatin structure and function during fertilization. It highlights the biological, assay and clinical uncertainties that require further research and ends with a series of 5 key recommendations.

  18. Sperm head structure of a murid rodent from southern Africa: the red veld rat Aethomys chrysophilus.

    PubMed

    Breed, W G; Cox, G A; Leigh, C M; Hawkins, P

    1988-02-01

    The morphology of spermatozoa from the red veld rat, Aethomys chrysophilus, of Southern Africa is described; two very different types were found, which came from animals from two separate, as-yet-undescribed, species. In individuals from South Africa the sperm head had a somewhat disc-shaped nucleus and a large acrosome with a huge apical segment that, during epididymal transit, changed in form from initially projecting anteriorly to a highly complex structure that was flexed caudad and lay alongside part of the rest of the sperm head. In addition, the chromatin generally appeared to be not fully condensed. Spermatozoa from animals collected in Malawi were very different in morphology and had a head with a typical apical hook, a perforatorium, fully condensed chromatin, and a 4-micron-long ventral spur. Its sperm tail was also significantly longer. The time of divergence of these two groups of animals from a common ancestor is not known, but the present results show that a considerable morphological change in the sperm nucleus, acrosome, and subacrosomal space can evolve even between two, presumably closely related, species.

  19. Sperm Morphology Assessment in Captive Neotropical Primates.

    PubMed

    Swanson, W F; Valle, R R; Carvalho, F M; Arakaki, P R; Rodas-Martínez, A Z; Muniz, Japc; García-Herreros, M

    2016-08-01

    The main objective of this study was to evaluate sperm morphology in four neotropical primate species to compare the sperm morphological traits and the sperm morphometric parameters as a basis for establishing normative sperm standards for each species. Data from 80 ejaculates collected from four primate species, Callithrix jacchus, Callimico goeldii, Alouatta caraya and Ateles geoffroyi, were analysed for detection of sperm morphological alterations using subjective World Health Organization (WHO-2010) standards and Sperm Deformity Index (SDI) criteria, objective computer-assisted sperm morphometry analysis (CASMA) and subpopulation sperm determination (SSD) methods. There were multiple differences (p < 0.01) observed among primate species in values obtained from WHO-2010, SDI, CASMA and SSD sperm analysis methods. In addition, multiple significant positive and negative correlations were observed between the sperm morphological traits (SDI, Sperm Deformity Index Head Defects, Sperm Deformity Index Midpiece Defects, Sperm Deformity Index Tail Defects, Normal Sperm, Head Defects, Midpiece Defects and Tail Defects) and the sperm morphometric parameters (SSD, Area (A), Perimeter (P), Length (L), Width (W), Ellipticity, Elongation and Rugosity) (p ≤ 0.046). In conclusion, our findings using different evaluation methods indicate that pronounced sperm morphological variation exists among these four neotropical primate species. Because of the strong relationship observed among morphological and morphometric parameters, these results suggest that application of objective analysis methods could substantially improve the reliability of comparative studies and help to establish valid normative sperm values for neotropical primates.

  20. Chromatin signatures of the Drosophila replication program.

    PubMed

    Eaton, Matthew L; Prinz, Joseph A; MacAlpine, Heather K; Tretyakov, George; Kharchenko, Peter V; MacAlpine, David M

    2011-02-01

    DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

  1. Human sperm chromosomes. Long-term effect of cancer treatment

    SciTech Connect

    Genesca, A.; Caballin, M.R.; Miro, R.; Benet, J.; Bonfill, X.; Egozcue, J. )

    1990-06-01

    The long-term cytogenetic effect of radio- or chemotherapy or both on male germ cells was evaluated by study of the chromosomal abnormalities in spermatozoa of four men treated for cancer 5-18 years earlier. The cytogenetic analysis of 422 sperm metaphases showed no differences in the aneuploidy rate. The incidence of structural chromosome aberrations was 14.0%, however, which is much higher than in controls. Thus, the high incidence of structurally aberrant spermatozoa observed in our long-term study indicates that antitumoral treatments affect stem-cell spermatogonia and that aberrant cells can survive germinal selection and produce abnormal spermatozoa.

  2. Chromatin endogenous cleavage and psoralen crosslinking assays to analyze rRNA gene chromatin in vivo.

    PubMed

    Griesenbeck, Joachim; Wittner, Manuel; Charton, Romain; Conconi, Antonio

    2012-01-01

    In eukaryotes, multiple copies of ribosomal RNA (rRNA) genes co-exist in two different chromatin states: actively transcribed (nucleosome depleted) chromatin, and nontranscribed (nucleosomal) chromatin. The presence of two rRNA gene populations compromises the interpretation of analyses obtained by the standard biochemical methods that are used to study chromatin structure (e.g., nuclease digestion and chromatin immunoprecipitation). Here, we provide a protocol to investigate the specific association of proteins with the two rRNA gene chromatin populations in vivo, using Saccharomyces cerevisiae as a model eukaryote.

  3. Role of Rb family in the epigenetic definition of chromatin.

    PubMed

    Gonzalo, Susana; Blasco, María A

    2005-06-01

    Epigenetic changes can influence a variety of cellular processes from regulation of gene transcription to proper chromosome segregation. The molecular activities that dictate the assembly, maintenance and regulation of chromatin structure are beginning to be identified. A recent study demonstrates that the Rb family of tumor suppressors plays a major role in global chromatin structure. In addition to the well-known function of Rb family inducing a repressive chromatin state around euchromatic promoters, Rb proteins have a direct role in the assembly of pericentric and telomeric heterochromatin domains. In particular, the Rb family maintains histone 4 lysine 20 tri-methylation (H4K20) at these constitutive heterochromatin domains. Lack of the Rb family results in decreased H4K20 tri-methylation, coincidental with chromosome segregation defects and abnormal telomere elongation, two processes frequently altered in human cancer. Maintenance of heterochromatic domains, such as those of centromeres and telomeres, may represent a novel tumor suppressor function for the Rb family by ensuing genomic stability.

  4. Alkali-labile sites in sperm cells from Sus and Ovis species.

    PubMed

    Cortés-Gutiérrez, Elva I; Dávila-Rodríguez, Martha I; López-Fernández, Carmen; Fernández, José Luis; Gosálvez, Jaime

    2008-06-01

    Constitutive alkali-labile sites (ALSs) have been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) in sperm cells from Sus domesticus (pig), Ovis gmelini musimon (mouflon) and Ovis aries (sheep). The results were compared with those obtained using leucocytes from the same species. Whole comparative genomic hybridization (W-CGH) showed that most of the constitutive ALSs in somatic and germ line cells in all species examined were constrained to particular repetitive satellite DNA sequences located in the pericentromeric constitutive heterochromatin of each chromosome. However, their relative abundance was different among cells of the same organism (leucocytes/sperm cells), and this trend was not maintained when the different species were compared. Thus, in mouflon, the density of ALSs in leucocytes when compared with that observed in sperm cells indicated abundance of the order of eight times less. In sheep, both leucocytes and sperm cells exhibited a large quantity of ALSs, being of the order of four times more abundant in sperm cells. In the pig genome, leucocytes showed a high abundance of ALSs (of the order of 12 times more that in sperm cells) but only involved the metacentric chromosomes of the karyotype. ALSs were not present in the acrocentric chromosomes. Contrary to mouflon and sheep, ALSs were relatively scarce in sperm cells from pig. These results suggest that ALSs are a transient structural feature in the cells of any organisms and point to a non-universal model of chromatin organization in sperm cells among mammals.

  5. Proteomics of a fuzzy organelle: interphase chromatin

    PubMed Central

    Kustatscher, Georg; Hégarat, Nadia; Wills, Karen L H; Furlan, Cristina; Bukowski-Wills, Jimi-Carlo; Hochegger, Helfrid; Rappsilber, Juri

    2014-01-01

    Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology. PMID:24534090

  6. Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition.

    PubMed

    Flyamer, Ilya M; Gassler, Johanna; Imakaev, Maxim; Brandão, Hugo B; Ulianov, Sergey V; Abdennur, Nezar; Razin, Sergey V; Mirny, Leonid A; Tachibana-Konwalski, Kikuë

    2017-04-06

    Chromatin is reprogrammed after fertilization to produce a totipotent zygote with the potential to generate a new organism. The maternal genome inherited from the oocyte and the paternal genome provided by sperm coexist as separate haploid nuclei in the zygote. How these two epigenetically distinct genomes are spatially organized is poorly understood. Existing chromosome conformation capture-based methods are not applicable to oocytes and zygotes owing to a paucity of material. To study three-dimensional chromatin organization in rare cell types, we developed a single-nucleus Hi-C (high-resolution chromosome conformation capture) protocol that provides greater than tenfold more contacts per cell than the previous method. Here we show that chromatin architecture is uniquely reorganized during the oocyte-to-zygote transition in mice and is distinct in paternal and maternal nuclei within single-cell zygotes. Features of genomic organization including compartments, topologically associating domains (TADs) and loops are present in individual oocytes when averaged over the genome, but the presence of each feature at a locus varies between cells. At the sub-megabase level, we observed stochastic clusters of contacts that can occur across TAD boundaries but average into TADs. Notably, we found that TADs and loops, but not compartments, are present in zygotic maternal chromatin, suggesting that these are generated by different mechanisms. Our results demonstrate that the global chromatin organization of zygote nuclei is fundamentally different from that of other interphase cells. An understanding of this zygotic chromatin 'ground state' could potentially provide insights into reprogramming cells to a state of totipotency.

  7. HDACi--targets beyond chromatin.

    PubMed

    Buchwald, Marc; Krämer, Oliver H; Heinzel, Thorsten

    2009-08-08

    Histone deacetylases (HDACs) play an important role in gene regulation. Inhibitors of HDACs (HDACi) are novel anti-cancer drugs, which induce histone (hyper-) acetylation and counteract aberrant gene repression. On the other hand, HDACi treatment can also result in decreased gene expression, and targeting HDACs affects more than chromatin. Recently, HDACi were shown to evoke non-histone protein acetylation, which can alter signaling networks relevant for tumorgenesis. Furthermore, HDACi can promote the degradation of (proto-) oncoproteins. Here, we summarize these findings and discuss how these substances could be beneficial for the treatment and prevention of human ailments, such as cancer and unbalanced immune functions.

  8. Preventive effect of Desferal on sperm motility and morphology.

    PubMed

    Nenkova, Galina; Stefanov, Rossen; Chervenkov, Mihail; Alexandrova, Albena

    2016-08-01

    Transition metal ions, mainly iron, are involved in the generation of highly reactive hydroxyl radicals, which are the most powerful inducers of oxidative damage to all biomolecules. The lipids in sperm membranes are highly susceptible to oxidation. Sperm lipid peroxidation (LPO) leads to decrease of motility and reduction of likelihood for sperm-oocyte fusion. The excess radical production may affect also the spermatozoa morphology. The aim of the present study was to investigate the effect of Desferal on the LPO, motility, and morphology of boar sperm subjected to oxidative stress. After collection, the ejaculates were equally separated and diluted in a commercial semen extender (experiment 1) or in physiological saline (experiment 2). The ejaculates of the 2 experiments were divided into aliquots, which were incubated with one of the following agents: FeSO4 (0.1mM), H2 O2 (0.5mM), or FeSO4  + H2 O2 (Fenton system), in the presence or absence of Desferal. The application of Desferal in the incubation medium had a protective effect against FeSO4  + H2 O2 -induced sperm damage, namely, decrease of LPO; decrease the quantity of immotile spermatozoa and decrease the number of morphological abnormalities, regardless of the used medium. In experiment 2, the presence of FeSO4 in the incubation medium induced LPO in the same range as the combination FeSO4  + H2 O2 , in which the effect was reduced by Desferal. Thus, the supplement of Desferal to media used for sperm storage and processing could be a useful tool for diminishing oxidative injury and improving the quality of the semen.

  9. Radiation-induced DNA content variability in mouse sperm

    SciTech Connect

    Pinkel, D.; Gledhill, B.L.; Van Dilla, M.A.; Lake, S.; Wyrobek, A.J.

    1983-09-01

    Mouse sperm collected from the cauda epididymidis 35 days after acute testicular X-ray exposure and fluorescently stained for DNA show dose-dependent increases in the coefficient of variation (CV) of flow cytometrically obtained fluorescence distributions. By comparing dose-response curves obtained with three protocols which overcome the optical and cytochemical difficulties of sperm measurement in different ways we conclude the response is due to X-ray-induced DNA content variability. In the range between 0 and 600 rad the dose dependence of the square of CV of the DNA content variability, delta CV2D, is described by delta CV2D . Bx + Cx2, with 0 less than or equal to B less than or equal to 0.23 X 10(-2) and C . (0.44 +/- 0.06) X 10(-4). The dose x is measured in rad and delta CVD is expressed in percent. Computer modeling of the shapes of the fluorescence distributions show that at 600 rad 30 to 40% of the sperm have abnormal DNA content. Some have errors as large as two whole chromosomes, but it is not clear whether they are due to whole chromosome nondisjunction or a finer fragmentation of the genome. Exposures to benzo(a)pyrene and mitomycin C cause no detectable DNA content variability. We conclude mouse sperm DNA content measurements are not sensitive to small amounts of aneuploidy and as such will only be useful in detecting agents that produce substantial DNA content variability. Another animal with a smaller number of chromosomes might be more favorable. These sperm measurement techniques may find additional application in other areas of reproductive biology, such as the determination of the relative numbers of X and Y chromosome-bearing sperm in semen that may be artificially enriched in one population.

  10. Oxidative Stress in Mouse Sperm Impairs Embryo Development, Fetal Growth and Alters Adiposity and Glucose Regulation in Female Offspring

    PubMed Central

    Lane, Michelle; McPherson, Nicole O.; Fullston, Tod; Spillane, Marni; Sandeman, Lauren; Kang, Wan Xian; Zander-Fox, Deirdre L.

    2014-01-01

    Paternal health cues are able to program the health of the next generation however the mechanism for this transmission is unknown. Reactive oxygen species (ROS) are increased in many paternal pathologies, some of which program offspring health, and are known to induce DNA damage and alter the methylation pattern of chromatin. We therefore investigated whether a chemically induced increase of ROS in sperm impairs embryo, pregnancy and offspring health. Mouse sperm was exposed to 1500 µM of hydrogen peroxide (H2O2), which induced oxidative damage, however did not affect sperm motility or the ability to bind and fertilize an oocyte. Sperm treated with H2O2 delayed on-time development of subsequent embryos, decreased the ratio of inner cell mass cells (ICM) in the resulting blastocyst and reduced implantation rates. Crown-rump length at day 18 of gestation was also reduced in offspring produced by H2O2 treated sperm. Female offspring from H2O2 treated sperm were smaller, became glucose intolerant and accumulated increased levels of adipose tissue compared to control female offspring. Interestingly male offspring phenotype was less severe with increases in fat depots only seen at 4 weeks of age, which was restored to that of control offspring later in life, demonstrating sex-specific impacts on offspring. This study implicates elevated sperm ROS concentrations, which are common to many paternal health pathologies, as a mediator of programming offspring for metabolic syndrome and obesity. PMID:25006800

  11. Genome-Wide Views of Chromatin Structure

    PubMed Central

    Rando, Oliver J.; Chang, Howard Y.

    2010-01-01

    Eukaryotic genomes are packaged into a nucleoprotein complex known as chromatin, which affects most processes that occur on DNA. Along with genetic and biochemical studies of resident chromatin proteins and their modifying enzymes, mapping of chromatin structure in vivo is one of the main pillars in our understanding of how chromatin relates to cellular processes. In this review, we discuss the use of genomic technologies to characterize chromatin structure in vivo, with a focus on data from budding yeast and humans. The picture emerging from these studies is the detailed chromatin structure of a typical gene, where the typical behavior gives insight into the mechanisms and deep rules that establish chromatin structure. Important deviation from the archetype is also observed, usually as a consequence of unique regulatory mechanisms at special genomic loci. Chromatin structure shows substantial conservation from yeast to humans, but mammalian chromatin has additional layers of complexity that likely relate to the requirements of multicellularity such as the need to establish faithful gene regulatory mechanisms for cell differentiation. PMID:19317649

  12. The Chd Family of Chromatin Remodelers

    PubMed Central

    Marfella, Concetta G.A.; Imbalzano, Anthony N.

    2007-01-01

    Chromatin remodeling enzymes contribute to the dynamic changes that occur in chromatin structure during cellular processes such as transcription, recombination, repair, and replication. Members of the chromodomain helicase DNA-binding (Chd) family of enzymes belong to the SNF2 superfamily of ATP-dependent chromatin remodelers. The Chd proteins are distinguished by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. Genetic, biochemical, and structural studies demonstrate that Chd proteins are important regulators of transcription and play critical roles during developmental processes. Numerous Chd proteins are also implicated in human disease. PMID:17350655

  13. Spermiogenesis and biflagellate spermatozoon of the teleost fish Lampanyctus crocodilus (Myctophiformes, Myctophidae): ultrastructure and characterisation of its sperm basic nuclear proteins.

    PubMed

    Ribes, E; Cheema, M; González-Romero, R; Lloris, D; Ausió, J; Saperas, N

    2015-08-01

    We undertook an ultrastructural study of the spermiogenesis of the lanternfish Lampanyctus crocodilus (Myctophiformes, Myctophidae) with special emphasis on the condensation of chromatin and the biochemical characterisation of its sperm nuclear basic proteins (SNBPs). The round head of the early spermatid of L. crocodilus develops into a curved conical-shaped head in the spermatozoon. Two flagella, present even in the spermatid, are inserted laterally at the convex side of the sperm head. Both flagella possess an axoneme with a 9 + 0 instead of the typical 9 + 2 axonemal structure. Mitochondria undergo a characteristic redistribution during spermiogenesis. A reduced number of them are present lying away from the centrioles at both ends of the concave side of the sperm head. During the chromatin condensation stages in spermiogenesis, fibrogranular structures with granules of 25 ± 5 and 50 ± 5 nm can be observed in the early spermatid and develop into larger granules of about 150 ± 50 nm in the middle spermatid. The latter granules coalesce during the transition to the advanced spermatid and spermatozoon giving rise to highly condensed chromatin in the sperm cell. Protamines are the main SNBPs associated with this chromatin; however, they are unusually large and correspond to the largest protamines described in fish to date. Small stoichiometric amounts of histones and other basic proteins coexist with these protamines in the spermatozoon.

  14. Effects of Cinnamon (C. zeylanicum) Bark Oil Against Taxanes-Induced Damages in Sperm Quality, Testicular and Epididymal Oxidant/Antioxidant Balance, Testicular Apoptosis, and Sperm DNA Integrity.

    PubMed

    Sariözkan, Serpil; Türk, Gaffari; Güvenç, Mehmet; Yüce, Abdurrauf; Özdamar, Saim; Cantürk, Fazile; Yay, Arzu Hanım

    2016-01-01

    The aim of this study was to investigate whether cinnamon bark oil (CBO) has protective effect on taxanes-induced adverse changes in sperm quality, testicular and epididymal oxidant/antioxidant balance, testicular apoptosis, and sperm DNA integrity. For this purpose, 88 adult male rats were equally divided into 8 groups: control, CBO, docetaxel (DTX), paclitaxel (PTX), DTX+PTX, DTX+CBO, PTX+CBO, and DTX+PTX+CBO. CBO was given by gavage daily for 10 weeks at the dose of 100 mg/kg. DTX and PTX were administered by intraperitoneal injection at the doses of 5 and 4 mg/kg/week, respectively, for 10 weeks. DTX+PTX and DTX+PTX+CBO groups were treated with DTX during first 5 weeks and PTX during next 5 weeks. DTX, PTX, and their mixed administrations caused significant decreases in absolute and relative weights of all reproductive organs, testosterone level, sperm motility, concentration, glutathione level, and catalase activity in testicular and epididymal tissues. They also significantly increased abnormal sperm rate, testicular and epididymal malondialdehyde level, apoptotic germ cell number, and sperm DNA fragmentation and significantly damaged the histological structure of testes. CBO consumption by DTX-, PTX-, and DTX+PTX-treated rats provided significant ameliorations in decreased relative weights of reproductive organs, decreased testosterone, decreased sperm quality, imbalanced oxidant/antioxidant system, increased apoptotic germ cell number, rate of sperm with fragmented DNA, and severity of testicular histopathological lesions induced by taxanes. In conclusion, taxanes cause impairments in sperm quality, testicular and epididymal oxidant/antioxidant balance, testicular histopathological structure, and sperm DNA integrity, and long-term CBO consumption protects male reproductive system of rats.

  15. Semen Quality and Sperm Function Loss by Hypercholesterolemic Diet Was Recovered by Addition of Olive Oil to Diet in Rabbit

    PubMed Central

    Romero, Aida A.; Funes, Abi K.; Cid-Barria, Macarena; Cabrillana, María E.; Monclus, María A.; Simón, Layla; Vicenti, Amanda E.; Fornés, Miguel W.

    2013-01-01

    Fat increment (0.05% cholesterol, chol) in standard diet promoted a significant increase in serum and sperm membrane chol, which ultimately altered membrane-coupled sperm specific functions: osmotic resistance, acrosomal reaction, and sperm capacitation in White New Zealand rabbits. These changes were also associated with a reduction in motility percentage and appearance of abnormal sperm morphology. The present study was carried out to evaluate the effect of dietary olive oil (OO, 7% v/w) administration to several male hypercholesterolemic rabbits (hypercholesterolemic rabbits, HCR) with altered fertility parameters. These HCR males were achieved by feeding normal rabbits with a high-fat diet (0.05% chol). HCR were associated with a modest non-significant increase in body weight (standard diet, 4.08±0.17 Kg, versus high-fat diet, 4.37±0.24 Kg). Hypercholesterolemic rabbits presented a marked decrease in semen volume, sperm cell count, and percentage of sperm motility, associated with a significant increase in sperm cell abnormalities. Moreover, sperm capacitation measured by the characteristic phosphorylated protein pattern in and induced acrosomal reaction were also altered suggesting sperm dysfunction. However, the administration of OO (for 16 weeks) to rabbits that were fed with 50% of the high-fat diet normalized serum chol. Curiously, OO supply succeeded to attenuate the seminal and sperm alterations observed in HCR group. Administration of OO alone did not cause any significant changes in above mentioned parameters. These data suggest that OO administration to HCR male rabbits recovers the loss of semen quality and sperm functionality. PMID:23326331

  16. Roberts syndrome: New evidence supporting an altered metaphase chromatin structure

    SciTech Connect

    Shang, X.M.; Schultz, E.L.; Tonk, V.

    1994-09-01

    Roberts syndrome is a rare autosomal recessive disease clinically manifested in the newborn by mental and growth retardation, tetraphocomelia, and a variety of craniofacial abnormalities. Cell lines derived from RS patients exhibit subtle mutagen hypersensitivity and cytogenetic abnormalities which include random chromosome loss and the splaying of heterochromatic chromosomal regions. The latter, typically detected on C-banded metaphases, has been used prenatally for the diagnosis of RS. To gain further insights into the RS defect, we have examined a number of parameters related to metaphase chromatin structure, with observations as follows. (1) The heterochromatic splaying associated with RS was found to be visible on G- as well as C-banded metaphases. (2) Quantitative evaluations using fluorescence image analysis revealed that RS metaphase chromosomes bind DAPI less efficiently than chromosomes from normal cells. (3) Denaturation of chromosomal DNA with either a C-banding procedure or 70% formamide at 70{degree}C each produced an aberrant hybridization pattern on RS chromosomes in FISH experiments employing biotinylated total human DNA as probe. (4) RS cells exhibited a >3-fold increase in sensitivity to VM-26, a potent inhibitor of topoisomerase II. Collectively, the aforementioned data support the notion that the primary defect in RS results in an altered metaphase chromatin structure.

  17. [Micro-insemination with intracytoplasmic sperm injection].

    PubMed

    Andersen, A G; Ziebe, S; Andersen, A N

    1996-11-18

    Intracytoplasmic sperm injection (ICSI) is now established in the treatment of infertility. Fertilization is achieved by microinjection of a single spermatozoon into the ooplasma. Oligoasthenoteratozoospermia is the main indication, but ICSI is also used in cases of failed fertilization after standard IVF, retrograde ejaculation and male immunological infertility. In obstructive azoospermia ICSI is performed after aspiration of epididymal or testicular spermatozoa. In some anejaculatoric men spermatozon can be obtained following penile vibration or electro-stimulation, but they often have poor motility and ICSI may be used for fertilization. ICSI may also be used after thawing of semen cryopreserved prior to treatment of a malignant disease. Since 1991 the ICSI technique has been improved, and today the pregnancy rates are at least as good as after standard IVF. So far, studies of the foetuses and children born after ICSI show that the number of malformations and abnormal karyotypes is within the range of the normal population.

  18. Applications of human sperm parameters for monitoring

    SciTech Connect

    Wyrobek, A.J.

    1985-11-01

    Very few studies of male reproductive hazards in the workplace are being done, and there are several underlying problems slowing the process in this infant field. First, the interaction of many diverse disciplines from management, labor, government, medicine, and science is often required. Outlooks and incentives are generally in conflict from the outset. Second, there is considerable complacency because no workplace agent as toxic as DBCP has been found. Third, there is as yet no requirement to assess the human male reproductive effects of a compound before large groups of men are permitted to work with it. Fourth, there is a poor opinion of the available epidemiological methods as indicators of abnormal reproductive outcome, because they are expensive, prone to confounding factors, and small numbers of exposed men (and pregnancies at risk) limit their sensitivity. Fifth, sperm tests have been criticized for their variability, subjectivity, and lack of standardization. 68 refs., 2 tabs.

  19. Nuclear Phosphoinositide Regulation of Chromatin.

    PubMed

    Hamann, Bree L; Blind, Raymond D

    2017-03-03

    Phospholipid signaling has clear connections to a wide array of cellular processes, particularly in gene expression and in controlling the chromatin biology of cells. However, most of the work elucidating how phospholipid signaling pathways contribute to cellular physiology have studied cytoplasmic membranes, while relatively little attention has been paid to the role of phospholipid signaling in the nucleus. Recent work from several labs has shown that nuclear phospholipid signaling can have important roles that are specific to this cellular compartment. This review focuses on the nuclear phospholipid functions and the activities of phospholipid signaling enzymes that regulate metazoan chromatin and gene expression. In particular, we highlight the roles that nuclear phosphoinositides play in several nuclear-driven physiological processes, such as differentiation, proliferation, and gene expression. Taken together, the recent discovery of several specifically nuclear phospholipid functions could have dramatic impact on our understanding of the fundamental mechanisms that enable tight control of cellular physiology. This article is protected by copyright. All rights reserved.

  20. Identification of lamin B–regulated chromatin regions based on chromatin landscapes

    PubMed Central

    Zheng, Xiaobin; Kim, Youngjo; Zheng, Yixian

    2015-01-01

    Lamins, the major structural components of the nuclear lamina (NL) found beneath the nuclear envelope, are known to interact with most of the nuclear peripheral chromatin in metazoan cells. Although NL–chromatin associations correlate with a repressive chromatin state, the role of lamins in tethering chromatin to NL and how such tether influences gene expression have remained challenging to decipher. Studies suggest that NL proteins regulate chromatin in a context-dependent manner. Therefore understanding the context of chromatin states based on genomic features, including chromatin–NL interactions, is important to the study of lamins and other NL proteins. By modeling genome organization based on combinatorial patterns of chromatin association with lamin B1, core histone modification, and core and linker histone occupancy, we report six distinct large chromatin landscapes, referred to as histone lamin landscapes (HiLands)-red (R), -orange (O), -yellow (Y), -green (G), -blue (B), and -purple (P), in mouse embryonic stem cells (mESCs). This HiLands model demarcates the previously mapped lamin-associated chromatin domains (LADs) into two HiLands, HiLands-B and HiLands-P, which are similar to facultative and constitutive heterochromatins, respectively. Deletion of B-type lamins in mESCs caused a reduced interaction between regions of HiLands-B and NL as measured by emerin–chromatin interaction. Our findings reveal the importance of analyzing specific chromatin types when studying the function of NL proteins in chromatin tether and regulation. PMID:25995381

  1. Fenitrothion alters sperm characteristics in rats: ameliorating effects of palm oil tocotrienol-rich fraction.

    PubMed

    Taib, Izatus Shima; Budin, Siti Balkis; Ghazali, Ahmad Rohi; Jayusman, Putri Ayu; Mohamed, Jamaludin

    2014-01-01

    Exposure to organophosphate insecticides such as fenitrothion (FNT) in agriculture and public health has been reported to affect sperm quality. Antioxidants may have a potential to reduce spermatotoxic effects induced by organophosphate. The present study was carried out to evaluate the effects of palm oil tocotrienol-rich fraction (TRF) in reducing the detrimental effects occurring in spermatozoa of FNT-treated rats. Adult male Sprague-Dawley rats were divided into four equal groups: a control group and groups of rats treated orally with palm oil TRF (200 mg/kg), FNT (20 mg/kg) and palm oil TRF (200 mg/kg) combined with FNT (20 mg/kg). The sperm characteristics, DNA damage, superoxide dismutase (SOD) activity, and levels of reduced glutathione (GSH), malondialdehyde (MDA), and protein carbonyl (PC) were evaluated. Supplementation with TRF attenuated the detrimental effects of FNT by significantly increasing the sperm counts, motility, and viability and decreased the abnormal sperm morphology. The SOD activity and GSH level were significantly increased, whereas the MDA and PC levels were significantly decreased in the TRF+FNT group compared with the rats receiving FNT alone. TRF significantly decreased the DNA damage in the sperm of FNT-treated rats. A significant correlation between abnormal sperm morphology and DNA damage was found in all groups. TRF showed the potential to reduce the detrimental effects occurring in spermatozoa of FNT-treated rats.

  2. Fenitrothion Alters Sperm Characteristics in Rats: Ameliorating Effects of Palm Oil Tocotrienol-Rich Fraction

    PubMed Central

    Taib, Izatus Shima; Budin, Siti Balkis; Ghazali, Ahmad Rohi; Jayusman,, Putri Ayu; Mohamed, Jamaludin

    2014-01-01

    Exposure to organophosphate insecticides such as fenitrothion (FNT) in agriculture and public health has been reported to affect sperm quality. Antioxidants may have a potential to reduce spermatotoxic effects induced by organophosphate. The present study was carried out to evaluate the effects of palm oil tocotrienol-rich fraction (TRF) in reducing the detrimental effects occurring in spermatozoa of FNT-treated rats. Adult male Sprague-Dawley rats were divided into four equal groups: a control group and groups of rats treated orally with palm oil TRF (200 mg/kg), FNT (20 mg/kg) and palm oil TRF (200 mg/kg) combined with FNT (20 mg/kg). The sperm characteristics, DNA damage, superoxide dismutase (SOD) activity, and levels of reduced glutathione (GSH), malondialdehyde (MDA), and protein carbonyl (PC) were evaluated. Supplementation with TRF attenuated the detrimental effects of FNT by significantly increasing the sperm counts, motility, and viability and decreased the abnormal sperm morphology. The SOD activity and GSH level were significantly increased, whereas the MDA and PC levels were significantly decreased in the TRF+FNT group compared with the rats receiving FNT alone. TRF significantly decreased the DNA damage in the sperm of FNT-treated rats. A significant correlation between abnormal sperm morphology and DNA damage was found in all groups. TRF showed the potential to reduce the detrimental effects occurring in spermatozoa of FNT-treated rats. PMID:25030881

  3. Sperm competition and sperm cooperation: the potential role of diploid and haploid expression.

    PubMed

    Immler, Simone

    2008-03-01

    Sperm competition is a powerful selective force driving the evolution of sperm shape and function. Recent findings suggest that sperm cooperation is a potential evolutionary response to sperm competition. Sperm cooperation may enhance the performance of the ejaculate increasing a male's chance to outcompete rival males in competition for fertilisation. Whether and how sperm cooperation may evolve is the focal point of this review. The relative importance of haploid and diploid gene expression for the evolution of sperm cooperation and the potential conflict of interest between (i) haploid sperm and diploid male and (ii) among sibling sperm, since sibling sperm only share an average of 50% of their genes in a diploid organism, are discussed. Furthermore, sperm cooperation is defined and the literature for empirical evidence of sperm cooperation is reviewed in light of the author's definitions.

  4. Sperm ubiquitination in epididymal feline semen.

    PubMed

    Vernocchi, Valentina; Morselli, Maria Giorgia; Varesi, Sara; Nonnis, Simona; Maffioli, Elisa; Negri, Armando; Tedeschi, Gabriella; Luvoni, Gaia Cecilia

    2014-09-01

    Ubiquitin is a 8.5-kDa peptide that tags other proteins for proteasomal degradation. It has been proposed that ubiquitination might be responsible for the elimination of defective spermatozoa during transit through the epididymis in humans and cattle, but its exact biological function in seminal plasma has not yet been clarified. In the domestic cat (Felis catus), the percentage of immature, unviable, and abnormal spermatozoa decreases during the epididymal transit, indicating the existence of a mechanism that removes defective spermatozoa. Magnetic cell separation techniques, based on the use of magnetic beads coated with anti-ubiquitin antibodies, may allow the selective capture of ubiquitinated spermatozoa from semen, thus contributing to the identification of a potential correlation between semen quality and ubiquitination process. Moreover, the selective identification of all the ubiquitinated proteins in different epididymal regions could give a better understanding of the ubiquitin role in feline sperm maturation. The aims of this study were as follows: (1) to verify the possibility of separating ubiquitinated spermatozoa with magnetic ubiquitin beads and identify the morphological and acrosomal differences between whole sample and unbound gametes, (2) to characterize all the ubiquitinated proteins in spermatozoa retrieved in the three epididymal regions by a proteomic approach. The data indicated the presence of ubiquitinated proteins in cat epididymal semen. However, a correlation between abnormal and ubiquitinated spermatozoa has not been found, and ubiquitin cannot be considered as a biomarker of quality of epididymal feline spermatozoa. To the author's knowledge, this is the first identification of all the ubiquitinated proteins of cat spermatozoa collected from different epididymal regions. The proteomic pattern allows a further characterization of cat epididymal semen and represents a contribute to a better understanding of the ubiquitin role in

  5. Potential changes in rat spermatogenesis and sperm parameters after inhalation of Boswellia papyrifera and Boswellia carterii incense.

    PubMed

    Ahmed, Mukhtar; Al-Daghri, Nasser; Alokail, Majed S; Hussain, Tajamul

    2013-02-28

    In this study the effect of Boswellia papyrifera (B. papyrifera) and Boswellia carterii (B. carterii) smoke exposure on spermatogenesis and sperm parameters in male albino rats was investigated. Rats (n = 11) were exposed daily in smoking chambers to smoke emanated by burning 4 g each of either B. papyrifera or B. carterii for 48 days. At the end of exposure duration rats were killed, and the testes were excised and analysed for histopathological and ultrastructural changes. Sperm analysis including total sperm count, motility, velocity and relative percentage of abnormal sperms were recorded. Rats exposed to B. papyrifera and B. carterii showed significant disturbances in spermatogenetic patterns and changes in sperm kinetics compared to unexposed rats. Atrophied seminiferous tubules with dynamic changes were also noticed. The boundaries of intercellular and intracellular vacuoles were seen in the Sertoli cells. Furthermore, in spermatids acrosomal vesicles were not fully formed. Degenerating spermatids were devoid of their nuclear membrane with electron dense matrix and vacuolization. Structural changes in Leydig cells were observed. Sperm analysis in exposed rats exhibited significant decrease in the sperm count, motility, speed and an increase in sperm anomalies when compare to controls. These findings demonstrate that the B. papyrifera and B. carterii smoke affects the process of spermatogenesis and sperm parameters and indicate the detrimental effects of these incense materials on human reproductive system.

  6. DNA flow cytometry of human spermatozoa: consistent stoichiometric staining of sperm DNA using a novel decondensation protocol.

    PubMed

    Kovács, Tamás; Békési, Gyöngyi; Fábián, Akos; Rákosy, Zsuzsa; Horváth, Gábor; Mátyus, László; Balázs, Margit; Jenei, Attila

    2008-10-01

    Rapid flow cytometric measurement of the frequency of aneuploid human sperms is in increasing demand but development of an exploitable method is hindered by difficulties of stoichiometric staining of sperm DNA. An aggressive decondensation protocol is needed after which cell integrity still remains intact. We used flow cytometry to examine the effect of lithium diiodosalicylate (LIS, chaotropic agent) on fluorescence intensity of propidium iodide-treated human spermatozoa from 10 normozoospermic men. When flow cytometric identification of diploid spermatozoa was achieved, validation was performed after sorting by three-color FISH. In contrast with the extremely variable histograms of nondecondensed sperms, consistent identification of haploid and diploid spermatozoa was possible if samples were decondensed with LIS prior to flow cytometry. A 76-fold enrichment of diploid sperms was observed in the sorted fractions by FISH. A significant correlation was found between the proportion of sorted cells and of diploid sperms by FISH. Application of LIS during the preparation of sperm for flow cytometry appears to ensure the stoichiometric staining of sperm DNA, making quantification of aneuploid sperm percentage possible. To our knowledge this is the first report in terms of separating spermatozoa with confirmedly abnormal chromosomal content. High correlation between the proportion of cells identified as having double DNA content by flow cytometry and diploid sperm by FISH allows rapid calculation of diploidy rate.

  7. Transcriptomes of isolated Oryza sativa gametes characterized by deep sequencing: evidence for distinct sex-dependent chromatin and epigenetic states before fertilization.

    PubMed

    Anderson, Sarah N; Johnson, Cameron S; Jones, Daniel S; Conrad, Liza J; Gou, Xiaoping; Russell, Scott D; Sundaresan, Venkatesan

    2013-12-01

    The formation of a zygote by the fusion of egg and sperm involves the two gametic transcriptomes. In flowering plants, the embryo sac embedded within the ovule contains the egg cell, whereas the pollen grain contains two sperm cells inside a supporting vegetative cell. The difficulties of collecting isolated gametes and consequent low recovery of RNA have restricted in-depth analysis of gametic transcriptomes in flowering plants. We isolated living egg cells, sperm cells and pollen vegetative cells from Oryza sativa (rice), and identified transcripts for approximately 36 000 genes by deep sequencing. The three transcriptomes are highly divergent, with about three-quarters of those genes differentially expressed in the different cell types. Distinctive expression profiles were observed for genes involved in chromatin conformation, including an unexpected expression in the sperm cell of genes associated with active chromatin. Furthermore, both the sperm cell and the pollen vegetative cell were deficient in expression of key RNAi components. Differences in gene expression were also observed for genes for hormonal signaling and cell cycle regulation. The egg cell and sperm cell transcriptomes reveal major differences in gene expression to be resolved in the zygote, including pathways affecting chromatin configuration, hormones and cell cycle. The sex-specific differences in the expression of RNAi components suggest that epigenetic silencing in the zygote might act predominantly through female-dependent pathways. More generally, this study provides a detailed gene expression landscape for flowering plant gametes, enabling the identification of specific gametic functions, and their contributions to zygote and seed development.

  8. Relative testis size and sperm morphometry across mammals: no evidence for an association between sperm competition and sperm length.

    PubMed Central

    Gage, Matthew J G; Freckleton, Robert P

    2003-01-01

    Understanding why there is extensive variation in sperm form and function across taxa has been a challenge because sperm are specialized cells operating at a microscopic level in a complex environment. This comparative study collates published data to determine whether the evolution of sperm morphometry (sperm total length and separate component dimensions) is associated with sperm competition (when different males' sperm mix and compete for a female's ova) across 83 mammalian species. We use relative testes mass as an indicator of the intensity of sperm competition across taxa: relative investment into testes is widely accepted to predict the level of sperm competition that a species or population endures. Although we found evidence for positive associations between relative testes mass (controlling for allometry) and sperm morphometry across 83 mammalian species, these relationships were phylogenetically dependent. When we appropriately controlled for phylogenetic association using multiple regression within a phylogenetic framework, there was no relationship between relative testes mass and sperm length across mammals. Furthermore, we found no evidence for associations between relative testes mass and sperm head, mid-piece or flagellar lengths, nor was there a relationship with mid-piece or mitochondrial volumes. Results, therefore, indicate that sperm competition does not select for longer or shorter sperm across mammals, and alternative forces selecting on sperm form and function are discussed. PMID:12769463

  9. Protective role of Hibiscus sabdariffa calyx extract against streptozotocin induced sperm damage in diabetic rats

    PubMed Central

    Idris, Muhd Hanis Md; Budin, Siti Balkis; Osman, Mohamad; Mohamed, Jamaludin

    2012-01-01

    Diabetes mellitus contributes to male sexual dysfunction and infertility by modulating oxidative damage. To date, a number of studies have demonstrated antioxidant properties of Hibiscus sabdariffa Linn. This study was designed to investigate the effects of H. sabdariffa UKMR-2 variety on sperm functioning of streptozotocin-induced diabetic rats. Male Sprague-Dawley rats were allotted into four groups, namely control group (C), H. sabdariffa extract (HSE) group, diabetes group (D) and diabetes plus HSE group (D+HSE). HSE (100 mg/ kg/body weight) was administered orally for 28 consecutive days. After 28-days of supplementation, the rats were sacrificed to obtain epididymal sperm. Administration of HSE significantly lowered the level of fasting blood glucose and increased plasma insulin level in D+HSE group as compared to D group (p<0.05). Sperm quality in the D+HSE group was improved with significantly higher sperm concentrations (p<0.05) and sperm motility (p<0.001) as well as lower percentage of sperm abnormality (p<0.05) as compared to the diabetic group. Plasma follicle-stimulating hormone (FSH) level was significantly elevated (p<0.05) in D+HSE group than in D group while no significant alteration in plasma testosterone and luteinizing hormone (LH) level were seen between groups. In conclusion, this study suggested that H. sabdariffa UKMR-2 variety has a potential protective role against diabetes-induced sperm damage. PMID:27847454

  10. Use of testicular sperm for ICSI in oligozoospermic couples: how far should we go?

    PubMed

    Zini, Armand; Bach, Phil V; Al-Malki, Ahmad H; Schlegel, Peter N

    2017-01-01

    In 1992 and subsequently, several reports indicated that ICSI was a successful technique to achieve clinical pregnancy and live birth using spermatozoa with severely impaired characteristics. The initial optimism over the ability of ICSI to overcome significant sperm abnormalities was later tempered by the findings of more recent publications suggesting that some sperm deficits may not be as effectively treated with ICSI. In search for effective treatment for couples with severe male factor, a number of small retrospective and prospective studies have reported high pregnancy and live birth rates using testicular sperm for men with necrozoospermia, cryptozoospermia and oligozoospermia with or without elevated sperm DNA damage. Although the data suggest that there may be some benefit in performing testicular sperm retrieval (TSR)-ICSI in select groups of non-azoospermic infertile men, there are potential risks involved with TSR. Clinicians should balance these risks prior to the recommendation of TSR-ICSI on the result of a semen analysis or sperm DNA test alone. Careful evaluation and management of male factor infertility is important. The use of TSR-ICSI in the absence of specific sperm DNA defects is still experimental.

  11. Laser light-scattering study of the toxic effects of methylmercury on sperm motility

    SciTech Connect

    Mohamed, M.K.; Lee, W.I.; Mottet, N.K.; Burbacher, T.M.

    1986-01-01

    An in vitro study was designed using the laser light-scattering technique to obtain further information on the dose-effect relationship of methylmercury on sperm motility. The technique provided a quantitative evaluation of sperm swimming speed. Semen samples were collected from normal male Macaca fascicularis monkeys by anal electroejaculation. Methylmercury was added to aliquots of sperm suspensions in BWW medium in doses of 10, 5, 2, and 1 ppm. After 3 hours, the relative speed was 35%, 59%, 69%, and 92% of the corresponding controls at doses of 10, 5, 2, and 1 ppm, respectively. The percentage of motile spermatozoa decreased significantly at 10 ppm. By microscopic observation abnormal motility was detected at 5 and 10 ppm, especially after 20 to 40 minutes. Head movement increased from side to side, and many spermatozoa developed coiled tails. The technique proved useful for defining the dose-effect relationship of methylmercury and sperm swimming speed.

  12. Comparison of three different techniques of human sperm DNA isolation for methylation assay.

    PubMed

    Yuan, Hong-fang; Kuete, Martin; Su, Li; Yang, Fan; Hu, Zhi-yong; Tian, Bo-zhen; Zhang, Hui-ping; Zhao, Kai

    2015-12-01

    Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method (method A), traditional phenol-chloroform method (method B), and TianGen kit method (method C). Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C (P<0.01). PCR results revealed that method A was more reliable in amplifying DEAD-box polypeptide 4 (DDX4) and copy number variations (CNVs) than methods B and C, which generated false-positive errors. The results of sperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reliable results and could be an optimal technique for extracting sperm DNA for methylation assay.

  13. Open chromatin reveals the functional maize genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Every cellular process mediated through nuclear DNA must contend with chromatin. As results from ENCODE show, open chromatin assays can efficiently integrate across diverse regulatory elements, revealing functional non-coding genome. In this study, we use a MNase hypersensitivity assay to discover o...

  14. Chromatin remodeling: nucleosomes bulging at the seams.

    PubMed

    Peterson, Craig L

    2002-04-02

    ATP-dependent chromatin remodeling enzymes, such as SWI/SNF, hydrolyze thousands of ATPs to regulate gene expression on chromatin fibers. Recent mechanistic studies suggest that these enzymes generate localized changes in DNA topology that drive formation of multiple, remodeled nucleosomal states.

  15. Chromatin roadblocks to reprogramming 50 years on.

    PubMed

    Skene, Peter J; Henikoff, Steven

    2012-10-29

    A half century after John Gurdon demonstrated nuclear reprogramming, for which he was awarded the 2012 Nobel Prize in Physiology or Medicine, his group provides insights into the molecular mechanisms whereby chromatin remodeling is required for nuclear reprogramming. Among the issues addressed in Gurdon's latest work are the chromatin impediments to artificially induced reprogramming, discovered by Shinya Yamanaka, who shared the award with Gurdon.

  16. A simple sperm nuclear vacuole assay with propidium iodide.

    PubMed

    Zhu, W-J; Li, J

    2015-09-01

    Our aim was to develop a new simple sperm nuclear vacuole assay (SNVA) with propidium iodide (PI) to determine the status of nuclear vacuole (NV) of individual spermatozoa. After PI staining, sperm nuclei were classified into the 14 categories according to both nuclear morphology and the status of NV. The incidence was 57.8% (range 28-84%) in fertile controls (n = 40), and 85.1% (range 67-99%) in men with varicocele (n = 40). In the fertile group, normal nuclear-shaped spermatozoa without NV or with one small NV located in the ante-nuclear region were significantly more in comparison with the varicocele group. In the varicocele group, abnormal nuclear-shaped spermatozoa with one large NV and with multiple NVs located in the ante-nuclear region were most frequent findings. Besides, spermatozoa with NVs in both ante- and post-nuclear regions in the varicocele group were significantly more than those in the fertile group. In both fertile and varicocele groups, normal or abnormal nuclear-shaped spermatozoa with one or more vacuoles only located in the post-nuclear region occurred sparingly. The SNVA provides a useful additional approach to identify the status of NV in human spermatozoa for diagnostic purposes. A good sperm sample would have more spermatozoa without NV or with one small NV located in the ante-nuclear region.

  17. TOPICAL REVIEW: The physics of chromatin

    NASA Astrophysics Data System (ADS)

    Schiessel, Helmut

    2003-05-01

    Recent progress has been made in the understanding of the physical properties of chromatin - the dense complex of DNA and histone proteins that occupies the nuclei of plant and animal cells. Here I will focus on the two lowest levels of the hierarchy of DNA folding into the chromatin complex. (i) The nucleosome, the chromatin repeating unit consisting of a globular aggregate of eight histone proteins with the DNA wrapped around it: its overcharging, the DNA unwrapping transition, the 'sliding' of the octamer along the DNA. (ii) The 30 nm chromatin fibre, the necklace-like structure of nucleosomes connected via linker DNA: its geometry, its mechanical properties under stretching and its response to changing ionic conditions. I will stress that chromatin combines two seemingly contradictory features: (1) high compaction of DNA within the nuclear envelope and, at the same time, (2) accessibility to genes, promoter regions and gene regulatory sequences.

  18. [Chromatin morphology and cytokinesis in pleurocapsalean cyanobacteria].

    PubMed

    Pinevich, A V; Gavrilova, O V; Averina, S G

    2007-01-01

    By means of differential interference contrast (DIC) and fluorescence microscopy, chromatin morphology and cytokinesis have been described in the cyanobacterium Pleurocapsa sp. CALU 1126 capable of multiple fission (multiple reproduction of the mother cell, the macrocyte, with formation of unique reproductive cells, the baeocytes). Two kinds of chromatin behavior have been revealed in the cell cycle: 1) the formation of numerous chromatin areas before their compartmentalization by multiple fission; 2) chromatin condensation in the phase of binary fission, and chromatin decondensation in growth period. The cytokinetic essence of multiple fission has been shown to consist of successive binary fissions of the macrocyte, while in between the mother cells (pre-baeocytes) do not grow.

  19. Interactions of transcription factors with chromatin.

    PubMed

    van Bakel, Harm

    2011-01-01

    Sequence-specific transcription factors (TFs) play a central role in regulating transcription initiation by directing the recruitment and activity of the general transcription machinery and accessory factors. It is now well established that many of the effects exerted by TFs in eukaryotes are mediated through interactions with a host of coregulators that modify the chromatin state, resulting in a more open (in case of activation) or closed conformation (in case of repression). The relationship between TFs and chromatin is a two-way street, however, as chromatin can in turn influence the recognition and binding of target sequences by TFs. The aim of this chapter is to highlight how this dynamic interplay between TF-directed remodelling of chromatin and chromatin-adjusted targeting of TF binding determines where and how transcription is initiated, and to what degree it is productive.

  20. Initiation of meiotic recombination in chromatin structure.

    PubMed

    Yamada, Takatomi; Ohta, Kunihiro

    2013-08-01

    Meiotic homologous recombination is markedly activated during meiotic prophase to play central roles in faithful chromosome segregation and conferring genetic diversity to gametes. It is initiated by programmed DNA double-strand breaks (DSBs) by the conserved protein Spo11, and preferentially occurs at discrete sites called hotspots. Since the functions of Spo11 are influenced by both of local chromatin at hotspots and higher-order chromosome structures, formation of meiotic DSBs is under regulation of chromatin structure. Therefore, investigating features and roles of meiotic chromatin is crucial to elucidate the in vivo mechanism of meiotic recombination initiation. Recent progress in genome-wide chromatin analyses tremendously improved our understanding on this point, but many critical questions are left unaddressed. In this review, we summarize current knowledge in the field, and also discuss the future problems that must be solved to understand the role of chromatin structure in meiotic recombination.

  1. Sperm in poor quality semen from bulls during heat stress have a lower affinity for binding hydrogen-3 heparin

    SciTech Connect

    Ax, R.L.; Gilbert, G.R.; Shook, G.E.

    1987-01-01

    Binding assays with (/sup 3/H) heparin were performed using spermatozoa collected prior to, during, and following summer heat stress to dairy bulls. Ejaculates collected in August 1983 after a period of ambient temperatures exceeding 29.4/sup 0/C exhibited a high frequency of abnormal sperm, and motility was reduced in some samples. Sperm in samples collected during heat stress possessed dissociation constants for binding (/sup 3/H) heparin ranging from 134.5 to 163.2 nmol. In contrast, sperm in semen collected prior to and after heat stress had significantly lower dissociation constants (higher affinity) for (/sup 3/H)heparin, 12.9 to 56.4 nmol. The number of binding sites for (/sup 3/H) heparin on sperm did not change among collection periods. It was concluded that the binding affinity for (/sup 3/H) heparin may reflect membrane integrity of bull sperm.

  2. Overlapping chromatin-remodeling systems collaborate genome wide at dynamic chromatin transitions.

    PubMed

    Morris, Stephanie A; Baek, Songjoon; Sung, Myong-Hee; John, Sam; Wiench, Malgorzata; Johnson, Thomas A; Schiltz, R Louis; Hager, Gordon L

    2014-01-01

    ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4 and Snf2h. The localization patterns of all three proteins substantially overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome wide and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes.

  3. Chromatin Dynamics of Circadian Transcription

    PubMed Central

    Aguilar-Arnal, Lorena; Sassone-Corsi, Paolo

    2015-01-01

    The molecular circadian clock orchestrates the daily cyclical expression of thousands of genes. Disruption of this transcriptional program leads to a variety of pathologies, including insomnia, depression and metabolic disorders. Circadian rhythms in gene expression rely on specific chromatin transitions which are ultimately coordinated by the molecular clock. As a consequence, a highly plastic and dynamic circadian epigenome can be delineated across different tissues and cell types. Intriguingly, genome topology appears to coordinate cyclic transcription at circadian interactomes, in which circadian genes are in physical contact within the cell nucleus in a time-specific manner. Moreover, the clock machinery shows functional interplays with key metabolic regulators, thereby connecting the circadian epigenome to cellular metabolism. Unraveling the molecular aspects of such interplays is likely to reveal new therapeutic strategies towards the treatment of metabolic disorders. PMID:27014564

  4. Sperm storage and viability in Photinus fireflies.

    PubMed

    Demary, Kristian C

    2005-07-01

    In many species females mate with and store sperm from multiple males, and some female insects have evolved multiple compartments for sperm storage. Sperm storage and sperm viability were investigated in two firefly species, Photinus greeni and P. ignitus, which differ in the morphology of the female reproductive tract. Although the primary spermatheca is similar in both species, P. greeni females have an additional, conspicuous outpocketing within the bursa copulatrix whose potential role in sperm storage was investigated in this study. An assay that distinguishes between live and dead sperm was used to examine sperm viability in male seminal vesicles and sperm storage sites within the female reproductive tract. For both Photinus species, sperm from male seminal vesicles showed significantly higher viability compared to sperm from the primary spermatheca of single mated females. In single mated P. greeni females, sperm taken from the channel outpocketing (secondary spermatheca) showed significantly higher viability compared to sperm from the primary spermatheca. This sperm viability difference was not evident in double mated females. There were no significant differences between P. greeni and P. ignitus females in the viability of sperm from the primary spermatheca. These studies contribute to our understanding of post-mating processes that may influence paternity success, and suggest that sexual conflict over control of fertilizations may occur in multiply mated firefly females.

  5. Morphological diversity of sperm: A mini review

    PubMed Central

    Prakash, Seppan; Prithiviraj, Elumalai; Suresh, Sekar; Lakshmi, Nagella Venkata; Ganesh, Mohanraj Karthik; Anuradha, Murugesan; Ganesh, Lakshmanan; Dinesh, Premavathy

    2014-01-01

    Sperms are highly specialized cells for delivering DNA from male to the ovum. Incredibly, wide degree of diversity in sperm morphology in their basic structures i.e. head, middle piece and tail is found across species. Differences in terms of overall size of the sperm, shape and number of sperm produced are also incredible. One of the key for this variations or diversity in sperm may be associated with female reproductive tract, sperm competition, testicular size and sperm size and number. Establishing a correlation between sperm morphology and factors influencing them is a phenomenal task. In this mini-review these associations and the anatomical and functional adaptations among different from of sperm cells that have evolved to optimize fertilization success are discussed. Nevertheless, explaining these morphological diversities in sperm cells is a challenging question and it seems that evolutionary biologists have only recently engaged in exploring its links and patterns. From the literatures it seems that there is no causal relationship between sperm size and testicular size, however, the accumulated knowledge do indicates evolution of sperm morphology across species has some associations with female reproductive tract, sperm competition and sperm size and number, however interpreting these results for phylogentic correlations should be approached with caution. PMID:24976817

  6. Computational strategies to address chromatin structure problems

    NASA Astrophysics Data System (ADS)

    Perišić, Ognjen; Schlick, Tamar

    2016-06-01

    While the genetic information is contained in double helical DNA, gene expression is a complex multilevel process that involves various functional units, from nucleosomes to fully formed chromatin fibers accompanied by a host of various chromatin binding enzymes. The chromatin fiber is a polymer composed of histone protein complexes upon which DNA wraps, like yarn upon many spools. The nature of chromatin structure has been an open question since the beginning of modern molecular biology. Many experiments have shown that the chromatin fiber is a highly dynamic entity with pronounced structural diversity that includes properties of idealized zig-zag and solenoid models, as well as other motifs. This diversity can produce a high packing ratio and thus inhibit access to a majority of the wound DNA. Despite much research, chromatin’s dynamic structure has not yet been fully described. Long stretches of chromatin fibers exhibit puzzling dynamic behavior that requires interpretation in the light of gene expression patterns in various tissue and organisms. The properties of chromatin fiber can be investigated with experimental techniques, like in vitro biochemistry, in vivo imagining, and high-throughput chromosome capture technology. Those techniques provide useful insights into the fiber’s structure and dynamics, but they are limited in resolution and scope, especially regarding compact fibers and chromosomes in the cellular milieu. Complementary but specialized modeling techniques are needed to handle large floppy polymers such as the chromatin fiber. In this review, we discuss current approaches in the chromatin structure field with an emphasis on modeling, such as molecular dynamics and coarse-grained computational approaches. Combinations of these computational techniques complement experiments and address many relevant biological problems, as we will illustrate with special focus on epigenetic modulation of chromatin structure.

  7. The effect of environmental exposure to pyrethroids and DNA damage in human sperm.

    PubMed

    Jurewicz, Joanna; Radwan, Michał; Wielgomas, Bartosz; Sobala, Wojciech; Piskunowicz, Marta; Radwan, Paweł; Bochenek, Michał; Hanke, Wojciech

    2015-01-01

    The present study was designed to investigate whether environmental exposure to pyrethroids was associated with sperm DNA damage. Between January 2008 and April 2011 286 men under 45 years of age with a normal sperm concentration of 15-300 10(6)/ml [WHO 2010] were recruited from an infertility clinic in Lodz, Poland. Participants were interviewed and provided urine, saliva, and semen samples. The pyrethroids metabolites: 3-phenoxybenzoic acid (3PBA), cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (CDCCA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (TDCCA), and cis-2,2-dibromovinyl-2,2-dimethylcyclopropane-carboxylic acid (DBCA) were analyzed in the urine using a validated gas chromatography ion-tap mass spectrometry method. Sperm DNA damage was assessed using a flow cytometry based on sperm chromatin structure assay (SCSA). A positive association was observed between CDCCA >50th percentile and the percentage of medium DNA fragmentation index (M DFI) and percentage of immature sperms (HDS) (p = 0.04, p = 0.04 respectively). The level of 3PBA >50th percentile in urine was positively related to the percentage of high DNA fragmentation index (H DFI) (p = 0.03). The TDCCA, DBCA levels, and the sum of pyrethroid metabolites were not associated with any sperm DNA damage measures. Our results suggest that environmental pyrethroid exposure may affect sperm DNA damage measures index indicated the reproductive effects of pyrethroid exposure on adult men. In view of the importance of human reproductive health and the widespread usage of pyrethroids, it is important to further investigate these correlations.

  8. Sperm-Egg Interaction: Evidence for Boar Sperm Plasma Membrane Receptors for Porcine Zona Pellucida

    NASA Astrophysics Data System (ADS)

    Peterson, Rudolph N.; Russell, Lonnie; Bundman, Donna; Freund, Matthew

    1980-01-01

    Freshly ejaculated, noncapacitated boar sperm bind rapidly and in large numbers to pig egg zona pellucida in vitro. In the present study, the number of sperm bound decreased sharply when sperm motility was lowered by energy poisons or by reducing the temperature. Highly motile sperm from humans, guinea pigs, and rats, added at concentrations ten times higher than control sperm, did not bind to the porcine zona. At the same high concentration, a small number of hamster and bull sperm bound to the zona. Binding of boar sperm to the zona pellucida was blocked almost completely by diluted whole antiserum to sperm plasma membranes and by univalent (Fab) antibody to these membranes. When antibody to sperm plasma membrane was first absorbed with plasma membrane vesicles, sperm binding was not inhibited. These results provide direct evidence for the existence of sperm plasma membrane receptors for the zona pellucida of the pig.

  9. 21 CFR 173.275 - Hydrogenated sperm oil.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... conditions: (a) The sperm oil is derived from rendering the fatty tissue of the sperm whale or is prepared by synthesis of fatty acids and fatty alcohols derived from the sperm whale. The sperm oil obtained...

  10. 21 CFR 173.275 - Hydrogenated sperm oil.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... conditions: (a) The sperm oil is derived from rendering the fatty tissue of the sperm whale or is prepared by synthesis of fatty acids and fatty alcohols derived from the sperm whale. The sperm oil obtained...

  11. 21 CFR 173.275 - Hydrogenated sperm oil.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... conditions: (a) The sperm oil is derived from rendering the fatty tissue of the sperm whale or is prepared by synthesis of fatty acids and fatty alcohols derived from the sperm whale. The sperm oil obtained...

  12. 21 CFR 173.275 - Hydrogenated sperm oil.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... conditions: (a) The sperm oil is derived from rendering the fatty tissue of the sperm whale or is prepared by synthesis of fatty acids and fatty alcohols derived from the sperm whale. The sperm oil obtained...

  13. Sperm competition games: the risk model can generate higher sperm allocation to virgin females.

    PubMed

    Ball, M A; Parker, G A

    2007-03-01

    We examine the risk model in sperm competition games for cases where female fertility increases significantly with sperm numbers (sperm limitation). Without sperm competition, sperm allocation increases with sperm limitation. We define 'average risk' as the probability q that females in the population mate twice, and 'perceived risk' as the information males gain about the sperm competition probability with individual females. If males obtain no information from individual females, sperm numbers increase with q unless sperm limitation is high and one of the two competing ejaculates is strongly disfavoured. If males can distinguish between virgin and mated females, greater sperm allocation to virgins is favoured by high sperm limitation, high q, and by the second male's ejaculate being disfavoured. With high sperm limitation, sperm allocation to virgins increases and to mated females decreases with q at high q levels. With perfect information about female mating pattern, sperm allocation (i) to virgins that will mate again exceeds that to mated females and to virgins that will mate only once, (ii) to virgins that mate only once exceeds that for mated females if q is high and there is high second male disadvantage and (iii) to each type of female can decrease with q if sperm limitation is high, although the average allocation increases at least across low q levels. In general, higher sperm allocation to virgins is favoured by: strong disadvantage to the second ejaculate, high sperm limitation, high average risk and increased information (perceived risk). These conditions may apply in a few species, especially spiders.

  14. Chromatin and microtubule morphology during the first cell cycle in bovine zygotes.

    PubMed

    Long, C R; Pinto-Correia, C; Duby, R T; Ponce de Leon, F A; Boland, M P; Roche, J F; Robl, J M

    1993-09-01

    Chromatin and microtubule configurations during the first cell cycle of bovine zygotes were analyzed by DNA staining and microtubule immunolocalization using an IVM/IVF system and oocytes matured and fertilized in vivo, in order to investigate the origin of the active centrosome and to characterize the nuclear and the cytoplasmic changes following bovine fertilization. Our results suggest that the paternal centrosome is active during early zygotic development, forming a conspicuous sperm aster soon after fertilization. We also report that polyspermy in bovine eggs, leads to the formation of numerous sperm asters with different degrees of association with the chromatin. The maternal structures in both monospermic and polyspermic zygotes can be lost or degenerate. Consequently, these cells may resume the first cell cycle as androgenotes, very often with several types of mitotic activity taking place in different regions of the cell cytoplasm at the same time. As indicated by a comparison of monospermic and polyspermic fertilization rates to rates of development, it is possible that some androgenetic embryos cleave and develop to the blastocyst stage.

  15. Absence of Peroxiredoxin 6 Amplifies the Effect of Oxidant Stress on Mobility and SCSA/CMA3 Defined Chromatin Quality and Impairs Fertilizing Ability of Mouse Spermatozoa1

    PubMed Central

    Ozkosem, Burak; Feinstein, Sheldon I.; Fisher, Aron B.; O'Flaherty, Cristian

    2016-01-01

    Oxidative stress, the imbalance between reactive oxygen species production and antioxidant defenses, is associated with male infertility. Peroxiredoxins (PRDXs) are antioxidant enzymes with a wide distribution in spermatozoa. PRDX6 is highly abundant and located in all subcellular compartments of the spermatozoon. Infertile men have lower levels of sperm PRDX6 associated with low sperm motility and high DNA damage. In order to better understand the role of PRDX6 in male reproduction, the aim of this study was to elucidate the impact of the lack of PRDX6 on male mouse fertility. Spermatozoa lacking PRDX6 showed significantly increased levels of cellular oxidative damage evidenced by high levels of lipid peroxidation, 8-hydroxy-deoxyguanosine (DNA oxidation), and protein oxidation (S-glutathionylation and carbonylation), lower sperm chromatin quality (high DNA fragmentation and low DNA compaction, due to low levels of protamination and a high percentage of free thiols), along with decreased sperm motility and impairment of capacitation as compared with wild-type (WT) spermatozoa. These manifestations of damage are exacerbated by tert-butyl hydroperoxide treatment in vivo. While WT males partially recovered the quality of their spermatozoa (in terms of motility and sperm DNA integrity), Prdx6−/− males showed higher levels of sperm damage (lower motility and chromatin integrity) 6 mo after the end of treatment. In conclusion, Prdx6−/− males are more vulnerable to oxidative stress than WT males, resulting in impairment of sperm quality and ability to fertilize the oocyte, compatible with the subfertility phenotype observed in these knockout mice. PMID:26792942

  16. Absence of beneficial effects on rabbit sperm cell cryopreservation by several antioxidant agents.

    PubMed

    Maya-Soriano, M J; Taberner, E; Sabés-Alsina, M; Piles, M; Lopez-Bejar, M

    2015-02-01

    The generation of reactive oxygen species associated with cryopreservation could be responsible for mammalian sperm damage and the limitable value of stored semen in artificial insemination. The aim of this study was to assess several antioxidant agents supplemented in a commercial freezing extender (Gent B®) in order to improve post-thaw rabbit sperm quality. Ejaculates of 26 New Zealand White rabbit bucks were collected, evaluated and frozen using a conventional protocol. Antioxidant agents were tested at different concentrations: bovine serum albumin (BSA; 5, 30 or 60 mg/ml), retinol (RO; 50, 100 or 200 μM) and retinyl (RI; 0.282 or 2.82 μg/ml). Per cent viability, morphological abnormalities and intact acrosomes were determined using eosin-nigrosin staining. Motility and progressivity were analyzed by computer-assisted sperm analysis (CASA). In general, all sperm quality parameters were negatively affected by the cryopreservation process, the largest effect seen was for total motility. The addition of antioxidant agents did not improve thaw sperm quality. Furthermore, for RI groups a significant decrease in sperm quality parameters was recorded. In conclusion, rabbit sperm quality is negatively affected by the cryopreservation process. To our knowledge this report is the first using these antioxidants to supplement rabbit freezing extender. BSA and RO at concentrations used in the study did not improve sperm quality parameters after thawing, whereas RI supplementation appeared to be toxic. More studies are required to find the appropriate antioxidants necessary and their most effective concentrations to improve rabbit post-thaw sperm quality.

  17. Effect of hyperthermia on replicating chromatin

    SciTech Connect

    Warters, R.L.; Roti Roti, J.L.

    1981-10-01

    The extent of heat-induced structural alterations in chromatin containing nascent (pulse-labeled) DNA was assayed using the enzyme micrococcal nuclease. The basic nucleosome structure in nascent and mature chromatin of S-phase cells appeared unaltered for up to 16 hr after exposure to hyperthermic temperatures as high as 48/sup 0/C for 15 min. However, the rate of nuclease digestion of DNA in both nascent and mature chromatin is inhibited following exposure to hyperthermic temperatures. In unheated cells, pulse-labeled nascent DNA matured into mature chromatin structure with a half-time of 2.5 min. The half-time for the maturation of pulse-labeled DNA from nascent into mature chromatin increased in a linear manner as a function of increasing temperature of exposure with constant heating time at temperatures above 43/sup 0/C. Both the reduced nuclease digestibility of nascent DNA and the increased time for chromatin structural changes could be due to the increased protein mass of chromatin following hyperthermia.

  18. Functional nonequivalence of sperm in Drosophila pseudoobscura.

    PubMed Central

    Snook, R R; Markow, T A; Karr, T L

    1994-01-01

    We report on a form of sperm polymorphism, termed polymegaly, that occurs in species of the Drosophila obscura group. Individual males of species in this group characteristically produce more than one discrete length of nucleated, motile sperm. Hypotheses suggested to explain the evolutionary significance of sperm polymorphism have been either nonadaptive or adaptive, with the latter focusing on sperm competition or nutrient provisioning. These hypotheses assume all sperm types fertilize eggs; however, no data have been gathered to test this assumption. We found that two size classes of sperm are produced and transferred to females in approximately equal numbers by the male; only long sperm persist in significant numbers in female sperm storage organs. Furthermore, we used a DNA-specific dye (bisbenzimide) and sperm-specific antibodies to ask if both sperm types fertilize eggs in Drosophila pseudoobscura. Confocal microscopy and immunofluorescent analyses of fertilized eggs using anti-sperm polyclonal antisera demonstrated that only long sperm participate in fertilization. These data falsify those hypotheses in which all sperm types are assumed to be functionally equivalent (fertilize eggs). Any remaining or new hypotheses for the evolutionary significance of polymegaly must incorporate these findings. Several new areas of research are suggested. Images PMID:7972038

  19. Activation of DNA damage response signaling by condensed chromatin.

    PubMed

    Burgess, Rebecca C; Burman, Bharat; Kruhlak, Michael J; Misteli, Tom

    2014-12-11

    The DNA damage response (DDR) occurs in the context of chromatin, and architectural features of chromatin have been implicated in DNA damage signaling and repair. Whereas a role of chromatin decondensation in the DDR is well established, we show here that chromatin condensation is integral to DDR signaling. We find that, in response to DNA damage chromatin regions transiently expand before undergoing extensive compaction. Using a protein-chromatin-tethering system to create defined chromatin domains, we show that interference with chromatin condensation results in failure to fully activate DDR. Conversely, forced induction of local chromatin condensation promotes ataxia telangiectasia mutated (ATM)- and ATR-dependent activation of upstream DDR signaling in a break-independent manner. Whereas persistent chromatin compaction enhanced upstream DDR signaling from irradiation-induced breaks, it reduced recovery and survival after damage. Our results demonstrate that chromatin condensation is sufficient for activation of DDR signaling and is an integral part of physiological DDR signaling.

  20. Parental origin of chromatin in human monopronuclear zygotes revealed by asymmetric histone methylation patterns, differs between IVF and ICSI.

    PubMed

    van der Heijden, G W; van den Berg, I M; Baart, E B; Derijck, A A H A; Martini, E; de Boer, P

    2009-01-01

    In mouse zygotes, many post-translational histone modifications are asymmetrically present in male and female pronuclei. We investigated whether this principle could be used to determine the genetic composition of monopronuclear human zygotes in conventional IVF and ICSI. First we determined whether male female asymmetry is conserved from mouse to human by staining polypronuclear zygotes with antibodies against a subset of histone N-tail post-translational modifications. To analyze human monopronuclear zygotes, a modification, H3K9me3, was selected that is present in the maternal chromatin. After IVF a total of 45 monopronuclear zygotes were obtained. In 39 (87%) of zygotes a nonuniform staining pattern was observed, proof of a bi-parental origin and assumed to result into a diploid conception. Two zygotes showed no staining for the modification, indicating that the single pronucleus was of paternal origin. Four zygotes contained only maternally derived chromatin. ICSI-derived monopronuclear zygotes (n = 33) could also be divided into three groups based on the staining pattern of their chromatin: (1) of maternal origin (n = 15), (2) of paternal origin (n = 8) or (3) consisting of two chromatin domains as dominating in IVF (n = 10). Our data show that monopronuclear zygotes originating from IVF generally arise through fusion of parental chromatin after sperm penetration. Monopronuclear zygotes derived from ICSI in most cases contain uni-parental chromatin. The fact that chromatin was of paternal origin in 24% of ICSI and in 4% of the IVF zygotes confirms earlier results obtained by FISH on cleavage stages. Our findings are of clinical importance in IVF and ICSI practice.

  1. Effect of sperm DNA fragmentation on clinical outcome of frozen-thawed embryo transfer and on blastocyst formation.

    PubMed

    Ni, Wuhua; Xiao, Shiquan; Qiu, Xiufang; Jin, Jianyuan; Pan, Chengshuang; Li, Yan; Fei, Qianjin; Yang, Xu; Zhang, Liya; Huang, Xuefeng

    2014-01-01

    During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ≤30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ≤30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles.

  2. The epididymal sperm viability, motility and DNA integrity in dead mice maintained at 4-6oC

    PubMed Central

    Golshan Iranpour, Farhad; Rezazadeh Valojerdi, Mojtaba

    2013-01-01

    Background: When male animals die, spermatozoa within the body of animal will be degenerated. Because of unique chromatin structure of sperm, maybe this degeneration is different from other cells. However there is not any research which considered directly the integrity of sperm DNA by keeping the cadaver in refrigerator. Objective: The aim of this study was to assess viability, total motility and DNA integrity of sperm cells after death. Materials and Methods:In this experimental study, 24 male Swiss white mice were killed by cervical dislocation and then kept in refrigerator (4-6oC) for up to 12 days. On the 0 (immediately after death as control group), 1st, 2nd, 3rd, 5th, 7th, 10th and the 12th days after death cauda epididymides were removed and squeezed in Ham’s F10 medium. The proportion of viable, motile and double stranded DNA spermatozoa was examined. Viability and DNA integrity of sperm cells were examined consecutively by eosin nigrosin and acridine orange stainings. Results:The data obtained from this study showed that viability and total motility of sperm cells were significantly decreased during 12 days after death (p<0.001). In contrast with viability and motility, DNA integrity was without significant changes (even 12 days after death). Conclusion:This study suggests that integrity of sperm DNA would not change even after 12 days after death if the cadaver kept in refrigerator. PMID:24639746

  3. Chromatin Fiber Dynamics under Tension and Torsion

    PubMed Central

    Lavelle, Christophe; Victor, Jean-Marc; Zlatanova, Jordanka

    2010-01-01

    Genetic and epigenetic information in eukaryotic cells is carried on chromosomes, basically consisting of large compact supercoiled chromatin fibers. Micromanipulations have recently led to great advances in the knowledge of the complex mechanisms underlying the regulation of DNA transaction events by nucleosome and chromatin structural changes. Indeed, magnetic and optical tweezers have allowed opportunities to handle single nucleosomal particles or nucleosomal arrays and measure their response to forces and torques, mimicking the molecular constraints imposed in vivo by various molecular motors acting on the DNA. These challenging technical approaches provide us with deeper understanding of the way chromatin dynamically packages our genome and participates in the regulation of cellular metabolism. PMID:20480035

  4. Nucleosome structure in chromatin from heated cells

    SciTech Connect

    Warters, R.L.; Roti Roti, J.L.; Winward, R.T.

    1980-12-01

    The effect of hyperthermia (40 to 80/sup 0/C) on the nucleosome structure of mammalian chromatin was determined using the enzyme micrococcal nuclease. At equivalent fractional DNA digestion it was found that neither the size of DNA nor the total fraction of cellular DNA associated with nucleosome structure is altered by heat exposure up to 48/sup 0/C for 30 min. It is proposed that this heat-induced reduction in the accessibility to nuclease attack of DNA in chromatin from heated cells is due to the increased protein mass associated with chromatin.

  5. reSETting chromatin during transcription elongation

    PubMed Central

    Smolle, Michaela; Workman, Jerry L.; Venkatesh, Swaminathan

    2013-01-01

    Maintenance of ordered chromatin structure over the body of genes is vital for the regulation of transcription. Increased access to the underlying DNA sequence results in the recruitment of RNA polymerase II to inappropriate, promoter-like sites within genes, resulting in unfettered transcription. Two new papers show how the Set2-mediated methylation of histone H3 on Lys36 (H3K36me) maintains chromatin structure by limiting histone dynamics over gene bodies, either by recruiting chromatin remodelers that preserve ordered nucleosomal distribution or by lowering the binding affinity of histone chaperones for histones, preventing their removal. PMID:23257840

  6. Unraveling chromatin structure using magnetic tweezers

    NASA Astrophysics Data System (ADS)

    van Noort, John

    2010-03-01

    The compact, yet dynamic organization of chromatin plays an essential role in regulating gene expression. Although the static structure of chromatin fibers has been studied extensively, the controversy about the higher order folding remains. The compaction of eukaryotic DNA into chromatin has been implicated in the regulation of all DNA processes. To understand the relation between gene regulation and chromatin structure it is essential to uncover the mechanisms by which chromatin fibers fold and unfold. We used magnetic tweezers to probe the mechanical properties of individual nucleosomes and chromatin fibers consisting of a single, well-defined array of 25 nucleosomes. From these studies five major features appeared upon forced extension of chromatin fibers: the elastic stretching of chromatin's higher order structure, the breaking of internucleosomal contacts, unwrapping of the first turn of DNA, unwrapping of the second turn of DNA, and the dissociation of histone octamers. These events occur sequentially at the increasing force. Neighboring nucleosomes stabilize DNA folding into a nucleosome relative to isolated nucleosomes. When an array of nucleosomes is folded into a 30 nm fiber, representing the first level of chromatin condensation, the fiber stretched like a Hookian spring at forces up to 4 pN. Together with a nucleosome-nucleosome stacking energy of 14 kT this points to a solenoid as the underlying topology of the 30 nm fiber. Surprisingly, linker histones do not affect the length or stiffness of the fibers, but stabilize fiber folding up to forces of 7 pN. The stiffness of the folded chromatin fiber points at histone tails that mediate nucleosome stacking. Fibers with a nucleosome repeat length of 167 bp instead of 197 bp are significantly stiffer, consistent with a two-start helical arrangement. The extensive thermal breathing of the chromatin fiber that is a consequence of the observed high compliance provides a structural basis for understanding the

  7. Chromatin targeting drugs in cancer and immunity.

    PubMed

    Prinjha, Rab; Tarakhovsky, Alexander

    2013-08-15

    Recent advances in the enzymology of transcription and chromatin regulation have led to the discovery of proteins that play a prominent role in cell differentiation and the maintenance of specialized cell functions. Knowledge about post-synthetic DNA and histone modifications as well as information about the rules that guide the formation of multimolecular chromatin-bound complexes have helped to delineate gene-regulating pathways and describe how these pathways are altered in various pathological conditions. The present review focuses on the emerging area of therapeutic interference with chromatin function for the purpose of cancer treatment and immunomodulation.

  8. No evidence of sperm conjugate formation in an Australian mouse bearing sperm with three hooks

    PubMed Central

    Firman, Renée C; Bentley, Blair; Bowman, Faye; Marchant, Fernando García-Solís; Parthenay, Jahmila; Sawyer, Jessica; Stewart, Tom; O'Shea, James E

    2013-01-01

    Sperm conjugation occurs when two or more sperm physically unite for motility or transport through the female reproductive tract. In many muroid rodent species, sperm conjugates have been shown to form by a single, conspicuous apical hook located on the sperm head. These sperm “trains” have been reported to be highly variable in size and, despite all the heads pointing in roughly the same direction, exhibit a relatively disordered arrangement. In some species, sperm “trains” have been shown to enhance sperm swimming speed, and thus have been suggested to be advantageous in sperm competition. Here, we assessed the behavior of sperm in the sandy inland mouse (Pseudomys hermannsburgensis), a muroid rodent that bears sperm with three apical hooks. First, we accrued genetic evidence of multiple paternity within “wild” litters to unequivocally show that sperm competition does occur in this species. Following this we utilized both in vitro and in vivo methodologies to determine whether sandy inland mouse sperm conjugate to form motile trains. Our observations of in vitro preparations of active sperm revealed that sandy inland mouse sperm exhibit rapid, progressive motility as individual cells only. Similarly, histological sections of the reproductive tracts of mated females revealed no in vivo evidence of sperm conjugate formation. We conclude that the unique, three-hooked morphology of the sandy inland mouse sperm does not facilitate the formation of motile conjugates, and discuss our findings in relation to the different hypotheses for the evolution of the muroid rodent hook/s. PMID:23919134

  9. Paternal poly (ADP-ribose) metabolism modulates retention of inheritable sperm histones and early embryonic gene expression.

    PubMed

    Ihara, Motomasa; Meyer-Ficca, Mirella L; Leu, N Adrian; Rao, Shilpa; Li, Fan; Gregory, Brian D; Zalenskaya, Irina A; Schultz, Richard M; Meyer, Ralph G

    2014-05-01

    To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo.

  10. Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.

    PubMed

    Cissen, Maartje; Wely, Madelon van; Scholten, Irma; Mansell, Steven; Bruin, Jan Peter de; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert

    2016-01-01

    Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the

  11. Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis

    PubMed Central

    Cissen, Maartje; van Wely, Madelon; Scholten, Irma; Mansell, Steven; de Bruin, Jan Peter; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert

    2016-01-01

    Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the

  12. Neutron scatter studies of chromatin structures related to function

    SciTech Connect

    Not Available

    1990-01-01

    In the Progress Report for last year (7-1-88 to 6-30-89) we proposed to complete the following experiments: (1) Structure of TFIIIA/DNA complex, (2) Effect of histone acetylation on nucleosome structure, and (3) Location of lysine rich histone H5 on the nucleosome. Our major source of neutrons is LANSCE, LANL. However, for the period of this report LANSCE has been down between cycles of operation. Continuing neutron scatter studies have been carried out at the Institute Laue Langevin, Grenoble, France, on the trimmed nucleosome core particles. X-ray scatter studies have been carried out at DESY, Hamburg on the histone octamer and trimmed octamer. X-ray scatter studies have been performed also at LANL on proposed objectives. We have continued with the following of our research program; (i) assembly of fully characterized nucleosomes; (ii) effect of histone acetylation on nucleosomes; (iii) effect of full acetylation of H3 and H4 on nucleosome DNA linking number; (iv) assembly and characterization of defined minichromosomes; (v) neutron and X-ray scatter of the histone octamer and trimmed octamer; (vi) structural studies of human sperm chromatin, histones and protamines. 5 refs.

  13. Chromatin dynamics during DNA replication

    PubMed Central

    Bar-Ziv, Raz; Voichek, Yoav; Barkai, Naama

    2016-01-01

    Chromatin is composed of DNA and histones, which provide a unified platform for regulating DNA-related processes, mostly through their post-translational modification. During DNA replication, histone arrangement is perturbed, first to allow progression of DNA polymerase and then during repackaging of the replicated DNA. To study how DNA replication influences the pattern of histone modification, we followed the cell-cycle dynamics of 10 histone marks in budding yeast. We find that histones deposited on newly replicated DNA are modified at different rates: While some marks appear immediately upon replication (e.g., H4K16ac, H3K4me1), others increase with transcription-dependent delays (e.g., H3K4me3, H3K36me3). Notably, H3K9ac was deposited as a wave preceding the replication fork by ∼5–6 kb. This replication-guided H3K9ac was fully dependent on the acetyltransferase Rtt109, while expression-guided H3K9ac was deposited by Gcn5. Further, topoisomerase depletion intensified H3K9ac in front of the replication fork and in sites where RNA polymerase II was trapped, suggesting supercoiling stresses trigger H3K9 acetylation. Our results assign complementary roles for DNA replication and gene expression in defining the pattern of histone modification. PMID:27225843

  14. Centromeric chromatin in fission yeast.

    PubMed

    Partridge, Janet F

    2008-05-01

    A fundamental requirement for life is the ability of cells to divide properly and to pass on to their daughters a full complement of genetic material. The centromere of the chromosome is essential for this process, as it provides the DNA sequences on which the kinetochore (the proteinaceous structure that links centromeric DNA to the spindle microtubules) assembles to allow segregation of the chromosomes during mitosis. It has long been recognized that kinetochore assembly is subject to epigenetic control, and deciphering how centromeres promote faithful chromosome segregation provides a fascinating intellectual challenge. This challenge is made more difficult by the scale and complexity of DNA sequences in metazoan centromeres, thus much research has focused on dissecting centromere function in the single celled eukaryotic yeasts. Interestingly, in spite of similarities in the genome size of budding and fission yeasts, they seem to have adopted some striking differences in their strategy for passing on their chromosomes. Budding yeast have "point" centromeres, where a 125 base sequence is sufficient for mitotic propagation, whereas fission yeast centromeres are more reminiscent of the large repetitive centromeres of metazoans. In addition, the centromeric heterochromatin which coats centromeric domains of fission yeast and metazoan centromeres and is critical for their function, is largely absent from budding yeast centromeres. This review focuses on the assembly and maintenance of centromeric chromatin in the fission yeast.

  15. Transcription of nucleosomes from human chromatin.

    PubMed Central

    Shaw, P A; Sahasrabuddhe, C G; Hodo, H G; Saunders, G F

    1978-01-01

    Nucleosomes (chromatin subunits) prepared by micrococcal nuclease digestion of human nuclei are similar in histone content but substantially reduced in non-histone proteins as compared to undigested chromatin. Chromatin transcription experiments indicate that the DNA in the nucleosomes is accessible to DNA-dependent RNA polymerase in vitro. The template capacities of chromatin and nucleosomes are 1.5 and 10%, respectively, relative to high molecular weight DNA, with intermediate values for oligonucleosomes. Three distinct sizes of transcripts, 150, 120 and 95 nucleotides in length, are obtained when nucleosomes are used as templates. However, when nucleosomal DNA is used as a template, the predominant size of transcripts is 150 nucleotides. When oligonucleosomes are used as templates longer transcripts are obtained. This indicates that RNA polymerase can transcribe the DNA contained in the nucleosomes. PMID:693325

  16. Predictive Computational Modeling of Chromatin Folding

    NASA Astrophysics Data System (ADS)

    di Pierro, Miichele; Zhang, Bin; Wolynes, Peter J.; Onuchic, Jose N.

    In vivo, the human genome folds into well-determined and conserved three-dimensional structures. The mechanism driving the folding process remains unknown. We report a theoretical model (MiChroM) for chromatin derived by using the maximum entropy principle. The proposed model allows Molecular Dynamics simulations of the genome using as input the classification of loci into chromatin types and the presence of binding sites of loop forming protein CTCF. The model was trained to reproduce the Hi-C map of chromosome 10 of human lymphoblastoid cells. With no additional tuning the model was able to predict accurately the Hi-C maps of chromosomes 1-22 for the same cell line. Simulations show unknotted chromosomes, phase separation of chromatin types and a preference of chromatin of type A to sit at the periphery of the chromosomes.

  17. Bioenergetics of Mammalian Sperm Capacitation

    PubMed Central

    Ferramosca, Alessandra; Zara, Vincenzo

    2014-01-01

    After ejaculation, the mammalian male gamete must undergo the capacitation process, which is a prerequisite for egg fertilization. The bioenergetics of sperm capacitation is poorly understood despite its fundamental role in sustaining the biochemical and molecular events occurring during gamete activation. Glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) are the two major metabolic pathways producing ATP which is the primary source of energy for spermatozoa. Since recent data suggest that spermatozoa have the ability to use different metabolic substrates, the main aim of this work is to present a broad overview of the current knowledge on the energy-producing metabolic pathways operating inside sperm mitochondria during capacitation in different mammalian species. Metabolism of glucose and of other energetic substrates, such as pyruvate, lactate, and citrate, is critically analyzed. Such knowledge, besides its obvious importance for basic science, could eventually translate into the development of novel strategies for treatment of male infertility, artificial reproduction, and sperm selection methods. PMID:24791005

  18. The making of abnormal spermatozoa: cellular and molecular mechanisms underlying pathological spermiogenesis.

    PubMed

    Chemes, Hector E; Rawe, Vanesa Y

    2010-09-01

    Fertilization in mammals occurs via a series of well-defined events in the secluded environment of the female reproductive tract. The mode of selection of the fertilizing spermatozoon nevertheless remains unknown. As has become evident during in vitro fertilization by sperm microinjection into the oocyte, abnormal spermatozoa can successfully fertilize oocytes. Under these extreme conditions, post-fertilization events, early embryonic development and implantation are significantly compromised indicating that the contribution of spermatozoa extends beyond sperm penetration. Microscopic identification of normal spermatozoa is a well-standardized procedure but insights into the mechanisms that lead to aberrant sperm differentiation and into the subcellular nature of sperm abnormalities have only recently begun to be obtained. The spermatozoon is the result of a complex development in which spermatid organelles give rise to various structural components with characteristic functions. Similar to other differentiated cells, the spermatozoon has a specific pathology that is most clearly identified by ultrastructural evaluation coupled with immunocytochemistry and molecular techniques. This multidisciplinary approach allows the precise characterization of sperm abnormalities, including structural, molecular and functional aspects. We summarize here studies of the physiopathology of spermiogenesis in two abnormal sperm phenotypes of infertile men: dysplasia of the fibrous sheath and acephalic spermatozoa/abnormal head-tail attachment. The characterization of the abnormalities of the tail cytoskeleton and centrioles has uncovered aspects of the subcellular basis of pathological spermiogenesis, has suggested experimental approaches to explore the nature of these anomalies and has opened the way for genetic studies that will ultimately lead to the design of the therapeutic tools of the future.

  19. Human sperm rheotaxis: a passive physical process

    PubMed Central

    Zhang, Zhuoran; Liu, Jun; Meriano, Jim; Ru, Changhai; Xie, Shaorong; Luo, Jun; Sun, Yu

    2016-01-01

    A long-standing question in natural reproduction is how mammalian sperm navigate inside female reproductive tract and finally reach the egg cell, or oocyte. Recently, fluid flow was proposed as a long–range guidance cue for sperm navigation. Coitus induces fluid flow from oviduct to uterus, and sperm align themselves against the flow direction and swim upstream, a phenomenon termed rheotaxis. Whether sperm rheotaxis is a passive process dominated by fluid mechanics, or sperm actively sense and adapt to fluid flow remains controversial. Here we report the first quantitative study of sperm flagellar motion during human sperm rheotaxis and provide direct evidence indicating that sperm rheotaxis is a passive process. Experimental results show that there is no significant difference in flagellar beating amplitude and asymmetry between rheotaxis-turning sperm and those sperm swimming freely in the absence of fluid flow. Additionally, fluorescence image tracking shows no Ca2+ influx during sperm rheotaxis turning, further suggesting there is no active signal transduction during human sperm rheotaxis. PMID:27005727

  20. SPERM COUNT DISTRIBUTIONS IN FERTILE MEN

    EPA Science Inventory

    Sperm concentration and count are often used as indicators of environmental impacts on male reproductive health. Existing clinical databases may be biased towards subfertile men with low sperm counts and less is known about expected sperm count distributions in cohorts of fertil...

  1. Human sperm rheotaxis: a passive physical process

    NASA Astrophysics Data System (ADS)

    Zhang, Zhuoran; Liu, Jun; Meriano, Jim; Ru, Changhai; Xie, Shaorong; Luo, Jun; Sun, Yu

    2016-03-01

    A long-standing question in natural reproduction is how mammalian sperm navigate inside female reproductive tract and finally reach the egg cell, or oocyte. Recently, fluid flow was proposed as a long–range guidance cue for sperm navigation. Coitus induces fluid flow from oviduct to uterus, and sperm align themselves against the flow direction and swim upstream, a phenomenon termed rheotaxis. Whether sperm rheotaxis is a passive process dominated by fluid mechanics, or sperm actively sense and adapt to fluid flow remains controversial. Here we report the first quantitative study of sperm flagellar motion during human sperm rheotaxis and provide direct evidence indicating that sperm rheotaxis is a passive process. Experimental results show that there is no significant difference in flagellar beating amplitude and asymmetry between rheotaxis-turning sperm and those sperm swimming freely in the absence of fluid flow. Additionally, fluorescence image tracking shows no Ca2+ influx during sperm rheotaxis turning, further suggesting there is no active signal transduction during human sperm rheotaxis.

  2. Sperm morphology of Muscidifurax uniraptor (Hymenoptera: Chalcidoidea: Pteromalidae).

    PubMed

    Santos, Helen Pinto; Barcellos, Marcelo Silva; Reis, Aline Beatriz; Dolder, Heidi; Lino-Neto, José

    2016-05-01

    Sperm morphology of the parasitoid Muscidifurax uniraptor was investigated under light and transmission electron microscopy. M. uniraptor sperm are filiform, spiraled, approximately 150 μm in length, with a distinctive head, hooded by an extracellular sheath and a flagellum. This extracellular layer, from which many filaments radiate, measures approximately 90 nm in thickness and covers a small acrosome and the anterior nuclear region. The acrosome is composed of an acrosomal vesicle and a perforatorium with its base inserted in the nuclear tip. The nucleus is filled with homogeneously compacted chromatin. The centriolar adjunct extends towards the anterior portion in a spiral around the nucleus for 3.5 μm in length. The two mitochondrial derivatives begin exactly at the centriole adjunct base and, in cross-section, have a circular shape with equal areas that are smaller than the axoneme diameter. It is coiled, with 9 + 9 + 2 microtubules and begins from the centriole, just below the nuclear base. The axoneme is connected to the mitochondrial derivatives by two small irregularly shaped masses. Between the derivatives and the axoneme, the 'center-flagellar material' is observed. Overall, these characteristics are recognized in other Chalcidoidea, especially in the eurytomids, but together they form a set of species-specific data.

  3. Nucleosome repeat lengths and columnar chromatin structure.

    PubMed

    Trifonov, Edward N

    2016-06-01

    Thorough quantitative study of nucleosome repeat length (NRL) distributions, conducted in 1992 by J. Widom, resulted in a striking observation that the linker lengths between the nucleosomes are quantized. Comparison of the NRL average values with the MNase cut distances predicted from the hypothetical columnar structure of chromatin (this work) shows a close correspondence between the two. This strongly suggests that the NRL distribution, actually, reflects the dominant role of columnar chromatin structure common for all eukaryotes.

  4. Chromatin fiber functional organization: Some plausible models

    NASA Astrophysics Data System (ADS)

    Lesne, A.; Victor, J.-M.

    2006-03-01

    We here present a modeling study of the chromatin fiber functional organization. Multi-scale modeling is required to unravel the complex interplay between the fiber and the DNA levels. It suggests plausible scenarios, including both physical and biological aspects, for fiber condensation, its targeted decompaction, and transcription regulation. We conclude that a major role of the chromatin fiber structure might be to endow DNA with allosteric potentialities and to control DNA transactions by an epigenetic tuning of its mechanical and topological constraints.

  5. Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells

    PubMed Central

    Martini, Emmanuelle; Roche, Danièle M.J.; Marheineke, Kathrin; Verreault, Alain; Almouzni, Geneviève

    1998-01-01

    The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair. PMID:9813080

  6. Transcriptional Coactivator PC4, a Chromatin-Associated Protein, Induces Chromatin Condensation▿ †

    PubMed Central

    Das, Chandrima; Hizume, Kohji; Batta, Kiran; Kumar, B. R. Prashanth; Gadad, Shrikanth S.; Ganguly, Semanti; Lorain, Stephanie; Verreault, Alain; Sadhale, Parag P.; Takeyasu, Kunio; Kundu, Tapas K.

    2006-01-01

    Human transcriptional coactivator PC4 is a highly abundant multifunctional protein which plays diverse important roles in cellular processes, including transcription, replication, and repair. It is also a unique activator of p53 function. Here we report that PC4 is a bona fide component of chromatin with distinct chromatin organization ability. PC4 is predominantly associated with the chromatin throughout the stages of cell cycle and is broadly distributed on the mitotic chromosome arms in a punctate manner except for the centromere. It selectively interacts with core histones H3 and H2B; this interaction is essential for PC4-mediated chromatin condensation, as demonstrated by micrococcal nuclease (MNase) accessibility assays, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images show that PC4 compacts the 100-kb reconstituted chromatin distinctly compared to the results seen with the linker histone H1. Silencing of PC4 expression in HeLa cells results in chromatin decompaction, as evidenced by the increase in MNase accessibility. Knocking down of PC4 up-regulates several genes, leading to the G2/M checkpoint arrest of cell cycle, which suggests its physiological role as a chromatin-compacting protein. These results establish PC4 as a new member of chromatin-associated protein family, which plays an important role in chromatin organization. PMID:16982701

  7. Dynamic chromatin modifications characterise the first cell cycle in mouse embryos.

    PubMed

    Santos, Fátima; Peters, Antoine H; Otte, Arie P; Reik, Wolf; Dean, Wendy

    2005-04-01

    On fertilisation, gametes undergo epigenetic reorganisation and re-establish totipotency. Here, we investigate links between chromatin remodelling and asymmetric maintenance of DNA methylation in the early mouse embryo. Using antibodies for lysine specific H3 methylation reveals that the male pronucleus is negative for di- and trimethyl H3-K9 yet the female is positive for these residues. However, the male is positive for monomethyl H3-K9 and H3-K27 and these signals increase during pronuclear maturation. Non-histone chromatin proteins of the Polycomb group are found in the paternal compartment as early as sperm decondensation. However, trimethyl H3-K27 is not observed in the male until the completion of DNA replication. Heterochromatin protein 1 beta (HP1beta) is abundant in the male pronucleus, despite the absence of di- and trimethyl H3-K9, and co-localises with monomethyl H3-K9. Recent evidence identifies monomethyl H3-K9 as the preferred substrate of Suvar39h, the histone methyl transferase (HMT) responsible for heterochromatic H3-K9 trimethylation. The association of HP1beta with monomethyl H3-K9 may assist in preventing further modification of H3-K9. Association of dimethylation but not trimethylation of H3-K9 with DNA methylation, in the female pronucleus, suggests a mechanistically significant link. These differences begin to provide a chromatin based explanation for paternal-specific active DNA demethylation and maternal specific protection in the mouse.

  8. Impact of cigarette smoking on histone (H2B) to protamine ratio in human spermatozoa and its relation to sperm parameters.

    PubMed

    Hamad, M F; Shelko, N; Kartarius, S; Montenarh, M; Hammadeh, M E

    2014-09-01

    Smoking is strongly associated with abnormalities in histone-to-protamine transition and with alteration of protamine expression in human spermatozoa. A proper protamine to histone ratio is, however, essential for sperm chromatin maturity and DNA integrity. Alterations in these sperm nuclear proteins were observed in infertile men. The present prospective study is aimed at evaluating the possible relationship among smoking