Orientation of bull sperms in static magnetic fields

The orientation of bull sperm cells in static magnetic fields was investigated by microscopic observation. The bull sperm, which has a very flat head, was fixed and its motion was stopped by glutaraldehyde. It was oriented with the whole body and the flat head perpendicular to the direction of the magnetic field. The diamagnetic cell components, such as the cell membrane, the DNA in the head, and the microtubule in the tail, were thought to contribute to this orientation, because bull sperm does not have paramagnetic components. For quantitative measurement of the orientation, the intensity of transmitted light through glutaraldehyde-fixed bull sperm suspension in a photometric cell was determined. The intensity changed slightly in proportion to the mean degree of orientation of the sperms. It increased sigmoidally depending on the intensity of the magnetic field and reached 100% at just below 1 T. The magnetic orientation is very strong in comparison to that of erythrocytes or platelets. By studying the response of the bull sperm to the magnetic field, it will be possible to understand its microstructure in more detail.     

Diurnal variation in sperm DNA fragmentation: analysis of 11,382 semen samples from two populations and in vivo animal experiments

ABSTRACT

Circadian rhythms have been found in some reproductive functions phenotypes but remain unclear for sperm DNA fragmentation index (DFI). The present study aims to investigate the diurnal variation of DFI in mice model and men sperm. Adult male mice were sacrificed for sperm DFI with Sperm Chromatin Structure Assay (SCSA) in 24 hours at 6 evenly distributed time points. A cosinor pattern of DFI was observed with a nadir at zeitgeber time 10 AM. In a community population with 630 semen samples collected between 8 AM and 20 PM, the temporal variation of DFI also fit a cosinor pattern with a ? 343° acrophase and a nadir at 11 AM (P = .031). In a reproductive-medical-center dataset of 10752 semen samples collected between 7 AM and 11 AM, the decreasing trend of DFI was also confirmed. For the males with multiple samples, intra-individual comparison between different timepoints was performed, and each consecutive hour after 7 AM was also associated with 2.5 (95% CI: ?1.0, 5.9)% lower DFI by SCSA or 4.9 (1.9, 7.8)% lower DFI by SCD. Our study reveals a daily diurnal variance in sperm DFI which may suggest a practical approach to get more qualified sperms for natural or assisted reproduction.

Abbreviations: BMI, Body mass index; DFI, DNA fragmentation index; MARHCS, Male Reproductive Health in the Chongqing College Students; RMC, Reproductive Medical Center; SCD, Sperm Chromatin Dispersion; SCSA, Sperm Chromatin Structure Assay.     

The alteration of PLCζ protein expression in unexplained infertile and asthenoteratozoospermic patients: A potential effect on sperm fertilization ability

Failed oocyte activation has been observed in unexplained infertile (UI) and asthenoteratozoospermic (AT) men. The deficiency of phospholipase C‐zeta (PLCζ) could be a possible reason for such failures and has not been studied yet. We investigated the expression and localization of PLCζ protein in the sperms of patients with UI and AT conditions. The relationships between PLCζ‐related parameters with male age, sperm characteristics, DNA integrity, and cellular maturity were assessed. Semen samples were collected from fertile (n = 40), UI (n = 40), and AT (n = 40) men. Subsequently, semen analysis, DNA fragmentation, hyaluronic acid‐binding ability, and PLCζ level along with its distribution were evaluated using computer‐assisted sperm analyzer, sperm chromatin structure assay (SCSA), hyaluronic acid‐binding assay (HBA), western blot analysis and immunofluorescence microscopy, respectively. Unlike SCSA, the values of HBA, and PLCζ expression were significantly reduced in UI and AT patients compared to fertile men, whereas no significant differences were observed among the experimental groups in terms of PLCζ localization patterns. The regression analysis also showed that HBA is the only variable associated with PLCζ levels. Furthermore, the correlation of male age with PLCζ localization in postacrosomal, equatorial, and acrosomal+postacrosomal+equatorial (A+PA+E) patterns, as well as the relation of normal morphology, with the (A+PA+E) pattern, remained in the regression model. Our findings indicated that reduced PLCζ level along with the increased DNA fragmentation and impaired maturation may be possible etiologies of decreased fertilization in the studied subjects.     

Orientation of paramecium swimming in a DC magnetic field

We found that a ciliated protozoan, Paramecium, swam perpendicular to a static (DC) magnetic field (0.68 T). The swimming orientation was similar even when the ionic current through the cell membrane disappeared after saponin treatment. To determine the diamagnetic anisotropy of intracellular organs, macronuclei, cilia, and secretory vesicles, trichocysts, were selectively isolated. Both cilia and trichocysts tended to align their long axis parallel to the magnetic field (0.78 T). Paramecium mutants that lack trichocysts also swam perpendicular to the magnetic field, although the proportion fraction was smaller than the normal population. Since large numbers of cilia and trichocysts are arranged at right angles to the long axis of the cell, the diamagnetic anisotropies of cilia and trichocysts cause the long axis of the cell to align perpendicular to the magnetic field. In contrast to the DC magnetic field, an alternative (AC) magnetic field (60 Hz, 0.65 T) had almost no effect on the swimming orientation of Paramecium.     

The staining pattern of human sperm with Diff Quik: relationship with sperm head morphology and a sperm chromatin structure assay (SCSA)

The dark staining of human sperm heads by Diff Quik is significantly correlated with abnormal sperm head morphology (r=0.51, p<0.0001), but is not associated with changes in sperm chromatin detected by a sperm chromatin structure assay (SCSA; r=0.18, p>0.09). Whilst valuable in the assessment of head morphology, it is concluded that Diff Quik staining is not a useful substitute for the SCSA to assess human sperm chromatin.     

The binding of exogenous DNA fragments to bovine spermatozoa

Abstract

The ability of mature, freeze‐thawed bovine sperm to bind exogenous end‐labelled or oligo‐labelled λ Hind III DNA restriction fragments was examined. Following 30 min. incubation of bovine sperm with P³² end‐labelled λ Hind III DNA and five washes with medium, approximately 5.8 ng DNA were bound to 10⁷ sperm. Agarose gel autoradiography revealed that all of the λ Hind III DNA bands were present following sperm washes except for the smaller 0.5 Kb and 0.125 Kb bands. Incubation of sperm with ³H oligo‐labelled λ Hind III DNA gave a much higher level of binding (138 ng/10⁷ sperm) than that found with end‐labelled DNA. This binding was entirely eliminated by DNase I. The separation of live and dead sperm fractions on Percoll gradients revealed that more oligo‐labelled λ Hind III DNA was found to be associated with the dead sperm fraction (31.2 ng/10⁷ sperm) rather than the live sperm fraction (2.7 ng/10⁷ sperm). Analysis of supravital stained, light microscopic autoradiographs confirmed that oligo‐labelled λ Hind III DNA bound to dead sperm in the post‐acrosomal region of the sperm head although other minor distribution patterns were observed.     

Magnetic resonance methods for studying intact spermatozoa

Motility is used as a routine parameter for assessing spermatozoa activity. The quality rating techniques adopted are based on electron or optical microscopy. However, these methods depend on gross structural and dynamical features of sperm cells and do not provide information on metabolic activity of intact cells. Lately, biochemical assays have become popular. Such methods are cumbersome and destroy the samples. Magnetic resonance methods offer a non-invasive method for studies on intact sperms. We have investigated respiration, maturation andin vitro capacitation of sperms from human ejaculates and sperms extracted from goat reproductive organ using electron spin resonance spin labelling and [³¹P] nuclear magnetic resonance methods. These studies clearly establish the advantages of magnetic resonance in studies related to metabolic activity of sperms.     

Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos) spermatozoa

The Cantabrian brown bear (Ursus arctos) is a highly endangered species in Spain and basic studies are necessary in order to bank its germplasm. Sperm heads are mainly made up of chromatin, thus their shape depends partly on chromatin structure. Thawed semen from 10 bears was used to analyze chromatin status by sperm chromatin structure assay (SCSA) and head morphometry by the computer-assisted sperm morphology assessment (CASMA) system. Morphometry was analyzed before and after freezing–thawing in order to evaluate the effects of cryopreservation on sperm heads. Each spermatozoon was measured for four primary parameters (length, L; width, W; area, A; perimeter, P) and derived parameters (ellipticity: L/W, circularity: 4πA/P², elongation: (L − W)/(L + W), regularity: πLW/4A). All the derived parameters significantly differed between bears. Likewise, cryopreservation affected head morphometry by reducing its size. Clustering based on morphometric parameters separated three subpopulations, one of them being significantly more influenced by the cryopreservation process. We obtained high correlations between head morphometry and SCSA parameters: standard deviation of DNA fragmentation index (SD-DFI) was correlated with perimeter and area (r = 0.75 and r = 0.62, respectively) and DFIm and DFIt (moderate and total DNA fragmentation index) were correlated with perimeter (r = 0.65 and r = 0.67, respectively). Nevertheless, classification of males according to SCSA or head morphometry did not completely agree so the two assays might explain male variability differently. We conclude that cryopreservation affected morphometry at least in a subset of spermatozoa. These results might improve future application of sperm banking techniques in this species.     

Analysis of anisotropic diamagnetic susceptibility of a bull sperm

Bull sperm and paramecium cilium were exposed to uniform static magnetic fields to observe their magnetic orientations and measure their anisotropic diamagnetic susceptibility (deltachi) for each. The prepared samples were whole bull sperm, bull sperm flat heads, and paramecium cilia, because bull sperm tails in a perfect condition could not be prepared. The whole bull sperm and the bull sperm heads became oriented perpendicular to the magnetic fields (1.7 Tesla maximum), while the paramecium cilia became parallel to the magnetic fields (8 Tesla maximum). A whole bull sperm, a bull sperm head, and a paramecium cilium were photometrically studied to obtain deltachi for each, which were estimated to be 1 x 10(-19), 3 x 10(-19), and 2 x 10(-20) J/T(2), respectively. deltachi of a sperm flagellum was estimated from the measured value of deltachi of the paramecium cilium, which agrees well with the difference between deltachi of the whole sperm and the sperm head. Additionally, this difference of deltachi almost coincides with the deltachi values calculated from deltachi of tubulin, as well. If the magnetic effect on biological systems is solved and the magnetic orientation correlates with it, deltachi will become the quantitative index of the effect.     

Large gradient high magnetic field affects the association of MACF1 with actin and microtubule cytoskeleton

The intense inhomogeneous magnetic fields acting on the diamagnetic materials naturally present in cells can generate strong magnetic forces. We have developed a superconducting magnet platform with large gradient high magnetic field (LG‐HMF), which can produce three magnetic force fields of ?1360, 0, and 1312 T²/m, and three corresponding apparent gravity levels, namely 0, 1, and 2‐g for diamagnetic materials. In this study, the effects of different magnetic force fields on osteoblast‐like cells (MG‐63 and MC3T3‐E1) viability, microtubule actin crosslinking factor 1 (MACF1) expression and its association with cytoskeleton were investigated. Results showed that cell viability increased to different degrees after exposure to 0 or 1‐g conditions for 24 h, but it decreased by about 30% under 2‐g conditions compared with control conditions. An increase in MACF1 expression at the RNA or protein level was observed in osteoblast‐like cells under the magnetic force field of ?1360 T²/m (0‐g) relative to 1312 T²/m (2‐g). Under control conditions, anti‐MACF1 staining was scattered in the cytoplasm and partially colocalized with actin filaments (AFs) or microtubules (MTs) in the majority of osteoblast‐like cells. Under 0‐g conditions, MACF1 labeling was concentrated at perinuclear region and colocalization was not apparent. The patterns of anti‐MACF1 labeling on MTs varied with MTs' changing under LG‐HMF environment. In conclusion, LG‐HMF affects osteoblast‐like cell viability, MACF1 distribution, expression, and its association with cytoskeleton to some extent. Bioelectromagnetics 30:545–555, 2009. © 2009 Wiley‐Liss, Inc.     

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 × 10⁶ cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 °C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.     

Evaluating human sperm DNA integrity: relationship between 8-hydroxydeoxyguanosine quantification and the sperm chromatin structure assay

In our work, we have used 8-hydroxy-deoxyguanosine (8-OH-dG), one of the major oxidative products of sperm DNA, in a population of patients consulting for infertility. We found an inverse relationship between sperm concentration and the log of the ratio of 8-OH-dG to dG (P < 0.01). On the same patients' sperm samples, the sperm chromatin structure assay (SCSA) was performed. An inverse relationship was observed between the DNA fragmentation index and sperm concentration (P < 0.001). There was also a positive relationship between SCSA and log 8-OH-dG/dG. This indicates that DNA fragmentation measured by the SCSA originates in part from oxidative products. In a few patients, antioxidant treatment decreased the DNA fragmentation index below the threshold of 30% that is crucial for subfertility.     

NMR approaches for monitoring domain orientations in calcium-binding proteins in solution using partial replacement of Ca by Tb

This work shows that the partial replacement of diamagnetic Ca²⁺ by paramagnetic Tb³⁺ in Ca²⁺/calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information. Species in which Tb³⁺ ions are bound to only one domain of calmodulin (the N-domain) and Ca²⁺ ions to the other (the C-domain) provide convenient systems for measuring these parameters. The nuclei in the C-domain experience the local magnetic field induced by the paramagnetic Tb³⁺ ions bound to the other domain at distances of over 40 Å from the Tb³⁺ ion, shifting the resonances for these nuclei. In addition, the Tb³⁺ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field. In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one-bond ¹⁵N-¹H splittings of up to 17 Hz in magnitude. The degree of alignment of the C-domain will also depend on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb³⁺ ions. Pseudocontact shifts for NH groups and ¹H-¹⁵N residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains.     

DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay

During the production of sex-sorted spermatozoa from bull semen, the cells are exposed to a number of potential hazards including: dilution, centrifugation, incubation, exposure to DNA stains and laser light. These factors may affect the survival capacity and fertilization potential of the sperm. The objective of this study was to determine whether sex-sorted bull spermatozoa have more DNA damage than sperm from conventional processed bull semen. Two methods were used to determine DNA integrity: the neutral Comet assay (NCA) and the sperm chromatin structure assay (SCSA). The NCA showed that the conventional samples had a higher tail moment (TM) (P < 0.017) than the sorted samples and that there was no difference between the samples in tail length (TL) (P = 0.36). The SCSA showed that the DNA fragmentation index (DFI) was higher for conventional than the sorted samples (P = 0.011), but the standard deviation of DFI (SD-DFI) was higher for the sorted samples (P < 0.001). We conclude that the NCA and SCSA can be used in assessing DNA integrity in bovine sperm and that cell sorting by flow cytometry improves the integrity of the sperm cell population. Additionally the results from the SCSA indicated that the sex-sorted sperm had less homogenous sperm chromatin. In the future assessment of sperm DNA integrity may be used to select bulls for sperm sex sorting and optimizing sperm sex sorting procedures.     

Genetic polymorphisms influence the susceptibility of men to sperm DNA damage associated with exposure to air pollution

The purpose of the present study was to investigate the impact of carcinogenic polycyclic aromatic hydrocarbons and volatile organic compounds on sperm quality in a group of city policemen in Prague during a period of increased concentrations of ambient air-pollutants (winter season) compared to a period of low exposure (spring). Polymorphisms in metabolic genes (CYP1A1, EPHX1, GSTM1, GSTP1, GSTT1), folic acid metabolism genes (MTR, MTHFR) and DNA repair genes (XRCC1, XPD6, XPD23, hOGG1) were evaluated in these men as potential modifiers of associations between air pollution exposure and changes in sperm quality. The study population was a group of 47 policemen working in the center of the city. Seasonal differences in exposure were verified by ambient and personal monitoring. Markers of sperm injury included semen volume, sperm concentration, sperm morphology, sperm motility, and sperm DNA damage measured with the sperm chromatin structure assay The sperm chromatin structure assay (SCSA) includes a measure of DNA damage called DNA Fragmentation Index (DFI). The % of cells with detectable DFI (detDFI) by this assay includes sperm with either medium or high DNA damage; the term hDFI is used to define the % of sperm with only high DNA damage. The assay also detects immature sperm defined by high density staining (HDS).No significant differences were found in any of the standard semen parameters between the sampling periods except for vitality of sperms. Both DFI and HDS were significantly higher in winter than in spring samples for all men and for non-smokers. At the bivariate level, significant associations between hDFI or detDFI and polymorphisms of the repair genes XRCC1, XPD6 and XPD23 were observed. In multivariate models, polymorphisms of the genes XPD6, XPD23 and CYP1A1MspI were associated with hDFI and HDS. Moreover, HDS was significantly associated with polymorphisms in GSTM1 gene.     

Efficient χ-tensor determination and NH assignment of paramagnetic proteins

Anisotropic magnetic susceptibility tensors χ of paramagnetic metal ions are manifested in pseudocontact shifts, residual dipolar couplings, and other paramagnetic observables that present valuable long-range information for structure determinations of protein-ligand complexes. A program was developed for automatic determination of the χ-tensor anisotropy parameters and amide resonance assignments in proteins labeled with paramagnetic metal ions. The program requires knowledge of the three-dimensional structure of the protein, the backbone resonance assignments of the diamagnetic protein, and a pair of 2D ¹⁵N-HSQC or 3D HNCO spectra recorded with and without paramagnetic metal ion. It allows the determination of reliable χ-tensor anisotropy parameters from 2D spectra of uniformly ¹⁵N-labeled proteins of fairly high molecular weight. Examples are shown for the 185-residue N-terminal domain of the subunit ε from E. coli DNA polymerase III in complex with the subunit θ and La³⁺ in its diamagnetic and Dy³⁺, Tb³⁺, and Er³⁺ in its paramagnetic form.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.The first two authors contributed equally to the project.     

Alterations of sperm DNA integrity during cryopreservation procedure and in vitro incubation of bull semen

An association between sperm DNA integrity and fertility was recently shown for frozen–thawed Norwegian Red (NRF) bull semen diluted in skimmed milk egg yolk (SMEY). In general the fertility of NRF cattle is high, however, in comparison with NRF semen in SMEY, NRF semen diluted in Tris EY based extenders has shown reduced fertility. The aim of the present study was to do a split-sample comparison of sperm DNA integrity of NRF bull semen (n = 20) in SMEY and Triladyl^® (Tris EY based) during routine cryopreservation procedure and during in vitro incubation of frozen–thawed semen in modified synthetic oviduct fluid (mSOF). In contrast to the high fertility of NRF cattle, Holstein cattle are experiencing a marked decline in fertility. Therefore, the present study also aimed to compare sperm DNA integrity of NRF (n = 20) and Holstein (n = 20) semen diluted in Triladyl^® during in vitro incubation. The sperm DNA integrity was measured by susceptibility to in situ acid induced denaturation by the Sperm chromatin structure assay (SCSA). Compared to initial values of frozen neat semen, an increase in DNA damage was observed after dilution and cooling (5 °C) and after freezing–thawing of NRF semen in SMEY, but only after freezing–thawing for NRF semen diluted in Triladyl^®. Sperm DNA damage of NRF semen increased during in vitro incubation in mSOF; the increase in percentage of spermatozoa with DNA damage was more prominent in SMEY than in Triladyl^®, while the degree of damage was higher in Triladyl^®, throughout the incubation period. However, while the correlation between DNA damage and sperm survival was negative in SMEY throughout the incubation period, a positive correlation was observed in Triladyl^® after 9 h of incubation, indicating a higher presence of DNA damage in the live sperm population. In comparison with Holstein spermatozoa, the sperm DNA integrity of NRF semen reflected a better ability to withstand alterations induced during in vitro incubation in mSOF. In conclusion, sperm DNA integrity of NRF bull semen was altered during the cryopreservation procedure and in vitro incubation in mSOF. Dilution in Triladyl^® maintained bull sperm DNA integrity better than dilution in SMEY. Furthermore, alterations in Holstein sperm DNA integrity was more pronounced during in vitro incubation in mSOF compared to NRF bull spermatozoa.     

The auto-orientation in high magnetic fields of oxidized cytochrome b562 as source of constraints for solution structure determination

¹⁵N-¹H ¹J couplings were measured at 500 MHz and 800 MHz for ¹⁵N enriched oxidized cytochrome b ₅₆₂ from E. coli. The magnetic field dependence of 70 ¹J values, which could be measured without signal overlap, shows that there is a molecular magnetic anisotropy which provides partial molecular orientation in the magnetic field and, consequently, residual dipolar couplings (rdc). The rdc were used as further constraints to improve the existing structure [Arnesano et al. (1999) Biochemistry, 38, 8657–8670] with a protocol which uses the rhombic anisotropy [Banci et al. (1998) J. Am. Chem. Soc., 120, 12903–12909]. The overall large molecular magnetic anisotropy has been found to be determined by both the low spin iron (III) and the four helix bundle structure magnetic susceptibility anisotropy contributions.     

Transient electric birefringence of the bacteriophages T3 and T7

Electric birefringence measurements of suspensions of T3 and T7 bacteriophages in 10^?2 M phosphate buffer, pH 6.9, show that there is a difference in their rotational diffusion coefficient. The values corrected to 25°C and water viscosity are D_25,w = 4630 ± 130 sec^?1 and D_25,w = 5290 ± 260 sec^?1 for T3 and T7, respectively. The value obtained from shell model calculations (according to Filson and Bloomfield) is D_25,w = 4500 ± 600 sec^?1. The apparent permanent dipole moments are 4.5 × 10^?26 C·m and 1.7 × 10^?26 C·m for T3 and T7, respectively. For both phage particles the intrinsic optical anisotropy is +7.2 × 10^?3. It is shown that this anisotropy is mainly due to the DNA molecule inside the head of the phage. Its positive value means that there exists an excess orientation of the DNA helix perpendicular to the symmetry axis of the particle. For T7 an unexpectedly large increase of Δn_s and K_sp occurs at a glycerol concentration of about 30% (v/v). This increase is interpreted as being caused by a change of the shape of the particle and/or a change in the secondary structure of the DNA inside the head of the bacteriophage.