Beneficial effects of hypotaurine supplementation in preparation and freezing media on human sperm cryo-capacitation and DNA quality

Abstract Background Although widely used, slow freezing considerably modifies the functions of human spermatozoa. Cryopreservation induces nuclear sperm alterations and cryo-capacitation, reducing the chances of pregnancy. Hypotaurine is naturally present in the male and female genital tracts and has capacitating, osmolytic and anti-oxidant properties. The analysis were performed on surplus semen of men with normal (n = 19) or abnormal (n = 14) sperm parameters. Spermatozoa were selected by density gradient centrifugation before slow freezing. For each sample, these steps were performed in parallel with (“H+” arm) or without (“H-” arm) hypotaurine supplementation. After thawing, we measured total and progressive mobility, vitality, acrosome integrity, markers of capacitation signaling pathway and nuclear quality. For the latter, we focused on sperm chromatin packaging, DNA fragmentation and the presence of vacuoles in the sperm nucleus. Results Post-thaw spermatozoa selected and frozen in the presence of hypotaurine had a higher vitality (+ 16.7%, p < 0.001), progressive and total motility (+ 39.9% and + 21.6% respectively, p < 0.005) than spermatozoa from the control “H-” arm. Hypotaurine also reduced the non-specific phosphorylation of the capacitation protein markers P110 and P80 (p < 0.01), indicating a decrease in cryo-capacitation. Hypotaurine supplementation reduced chromatin decondensation, measured by chromomycin A3 (− 16.1%, p < 0.05), DNA fragmentation (− 18.7%, p < 0.05) and nuclear vacuolization (− 20.8%, p < 0.05). Conclusion Our study is the first to demonstrate beneficial effects of hypotaurine supplementation in preparation and freezing procedures on human spermatozoa sperm fertilization capacity and nucleus quality. Hypotaurine supplementation limited cryo-capacitation, increased the proportion of live and progressively motile spermatozoa and reduces the percentage of spermatozoa showing chromatin decondensation, DNA fragmentation and nuclear vacuolation. Trial registration Clinical Trial, NCT04011813. Registered 19 May 2019 - Retrospectively registered.

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A High Resolution Cytochemical Study of Nuclear ATPase in Human Spermatozoa

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100116590 ◽

1975 ◽

Vol 33 ◽

pp. 594-595

Author(s):

A. Sosa ◽

L. Calzada

Keyword(s):

High Resolution ◽

Atpase Activity ◽

Nuclear Membrane ◽

Human Sperm ◽

Sperm Nucleus ◽

Human Spermatozoa ◽

Cytochemical Study ◽

Membrane Bound ◽

Sperm Nuclei ◽

Interchromatin Space

The dependence of nuclear metabolism on the function of the nuclear membrane is not well understood. Whether or not the function of the nuclear membrane is partial or totally responsible of the repressed template activity of human sperm nucleus has not at present been elucidated. One of the membrane-bound enzymatic activities which is concerned with the mechanisms whereby substances are thought to cross cell membranes is adenosintriphosphatase (ATPase). This prompted its characterization and distribution by high resolution photogrammetry on isolated human sperm nuclei. Isolated human spermatozoa nuclei were obtained as previously described. ATPase activity was demonstrated by the method of Wachstein and Meisel modified by Marchesi and Palade. ATPase activity was identified as dense and irregularly distributed granules confined to the internal leaflet of the nuclear membrane. Within the nucleus the appearance of the reaction product occurs as homogenous and dense precipitates in the interchromatin space.

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

Reproduction ◽

10.1530/rep-09-0021 ◽

2009 ◽

Vol 138 (2) ◽

pp. 267-278 ◽

Cited By ~ 32

Author(s):

Yeng Peng Zee ◽

Carmen López-Fernández ◽

F Arroyo ◽

Stephen D Johnston ◽

William V Holt ◽

...

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Dna Fragmentation ◽

Sperm Nucleus ◽

Severe Form ◽

Sperm Chromatin ◽

Dna Breaks ◽

Single Stranded Dna ◽

Double Stranded Dna ◽

Dispersion Test

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.

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Evaluation of normal morphology, DNA fragmentation, and hyaluronic acid binding ability of human spermatozoa after using four different commercial media for density gradient centrifugation

Clinical and Experimental Reproductive Medicine ◽

10.5653/cerm.2019.46.1.8 ◽

2019 ◽

Vol 46 (1) ◽

pp. 8-13 ◽

Cited By ~ 1

Author(s):

Dayong Lee ◽

Byung Chul Jee

Keyword(s):

Hyaluronic Acid ◽

Dna Fragmentation ◽

Density Gradient ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Human Spermatozoa ◽

Binding Ability ◽

Normal Morphology ◽

Commercial Media ◽

Hyaluronic Acid Binding

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Human sperm chromatin forms spatially restricted nucleosome domains consistent with programmed nucleosome positioning

10.1101/481028 ◽

2018 ◽

Author(s):

Mei-Zi Zhang ◽

Xiao-Min Cao ◽

Feng-Qin Xu ◽

Xiao-Wei Liang ◽

Long-Long Fu ◽

...

Keyword(s):

Small Sample Size ◽

Three Dimensional ◽

Human Sperm ◽

Nucleosome Positioning ◽

Sperm Nucleus ◽

Small Sample ◽

Sperm Chromatin ◽

Epigenetic Mark ◽

Structured Illumination ◽

Structured Illumination Microscopy

AbstractIn human sperm, a fraction of its chromatin retains nucleosomes that are positioned on specific sequences containing genes and regulatory units essential for embryonic development. This nucleosome positioning (NP) feature provides an inherited epigenetic mark for sperm. However, it is not known whether there is a structural constraint for these nucleosomes and, if so, how they are localized in a three-dimensional (3D) context of the sperm nucleus. In this study, we examine the 3D organization of sperm chromatin and specifically determine its 3D localization of nucleosomes using structured illumination microscopy. A fraction of the sperm chromatin form nucleosome domains (NDs), visible as microscopic puncta ranging from 40 μm to 700 μm in diameter, and these NDs are precisely localized in the postacrosome region (PAR), outside the sperm’s core chromatin. Further, NDs exist mainly in sperm from fertile men in a pilot survey with a small sample size. Together, this study uncovers a new spatially restricted sub-nuclear structure containing NDs that are consistent with NPs of the sperm, which might represent a novel mark for healthy sperm in human.

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255 SPERM NUCLEAR DECONDENSATION ABILITY OF IN VITRO MATURED CANINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab255 ◽

2011 ◽

Vol 23 (1) ◽

pp. 225

Author(s):

M. De los Reyes ◽

J. Vergara ◽

J. Palomino

Keyword(s):

Cumulus Cells ◽

Sperm Nucleus ◽

Culture Conditions ◽

Sperm Chromatin ◽

Chromatin Decondensation ◽

Chi Square ◽

Cytoplasmic Maturation ◽

Sperm Nuclei ◽

Nuclear Decondensation

The sperm chromatin decondensation occurs when a spermatozoon enters into an oocyte during fertilization, and the effectiveness of this process is connected to the grade of oocyte cytoplasmic maturation. In this study chromatin sperm decondensation was evaluated after IVF of canine oocytes matured in vitro (IVM), comparing different durations of maturation. Cumulus–oocytes complexes (COC) for IVM were obtained from bitch ovaries after ovariohysterectomy, selecting those COC with compact cumulus cells and a homogeneous dark cytoplasm. The COC were matured in vitro for 0, 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM HEPES, 10% FCS, 0.25 mM pyruvate, 10 IU mL–1 of hCG, 300 IU mL–1 of penicillin, and 20 mg mL–1 of streptomycin at 38.5°C and 5% CO2. Fresh ejaculates from 3 adult dogs were centrifuged, and the sperm pellet was resuspended in fert-TALP medium. In each replicate, 100-μL fert-TALP drops containing 10 to 12 IVM oocytes after each culture time were co-culture with 2.5 × 106 spermatozoa mL–1 for 24 h under culture conditions. Soon after co-culture, all oocytes were denuded from cumulus cells and fixed in 3% paraformaldehyde. The nuclear stage of the oocytes and the appearance of the sperm nucleus were determined by 4′,6-diamidino-2-phenylindole staining under a fluorescence inverted microscope. For each treatment, at least 4 replicates were performed, and the data were compared statistically by chi-square test, using InfoStat Professional Program. A total of 800 oocytes were evaluated, and the percentages of oocytes with sperm penetration were 58% (138/238), 61% (108/177), 72% (118/165), and 70% (153/220) at 0, 48, 72, and 96 h of IVM, respectively. The percentage of sperm nuclear decondensation at each time point significantly increased up to 72 h of culture, showing 12, 34, 81, and 85% of sperm nuclei deconsated, respectively. The percentages of nuclear maturation also increased (P ≤ 0.05) with time, showing 0, 8, 20, and 27% of oocytes at second metaphase (MII) stage at 0, 48, 72, and 96 h of culture. The percentage of MII stage was much lower than that of chromatin decondensation in all maturing groups. These results suggest that canine oocytes matured in vitro are able to decondense the sperm chromatin during IVF, and this ability increases up to 72 h of culture. Nevertheless, cytoplasmic maturation, as evaluated by sperm chromatin decondensation, in canine oocytes matured in vitro may not be completely connected with nuclear development. This work was supported by Grant FONDECYT 1080618.

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Sperm chromatin dispersion test in the assessment of DNA fragmentation and aneuploidy in human spermatozoa

Reproductive BioMedicine Online ◽

10.1016/j.rbmo.2011.01.012 ◽

2011 ◽

Vol 22 (5) ◽

pp. 428-436 ◽

Cited By ~ 22

Author(s):

A. Balasuriya ◽

B. Speyer ◽

P. Serhal ◽

A. Doshi ◽

J.C. Harper

Keyword(s):

Dna Fragmentation ◽

Human Spermatozoa ◽

Sperm Chromatin ◽

Dispersion Test ◽

Sperm Chromatin Dispersion Test

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Effect of temperature, nutrients, calcium, and cAMP on motility of human spermatozoa

AJP Cell Physiology ◽

10.1152/ajpcell.1982.242.5.c304 ◽

1982 ◽

Vol 242 (5) ◽

pp. C304-C311 ◽

Cited By ~ 13

Author(s):

F. K. Gorus ◽

R. Finsy ◽

D. G. Pipeleers

Keyword(s):

Seminal Fluid ◽

Gradient Centrifugation ◽

Cell Movement ◽

Human Sperm ◽

Correlation Spectroscopy ◽

Human Spermatozoa ◽

Effect Of Temperature ◽

Dependent Manner ◽

Calcium Dependent ◽

Progressive Motility

The motility of human spermatozoa and its regulation were examined on cells isolated from other seminal components and purified into fractions of uniform progressive motility. The percent motile cells and estimates of their translational speed were determined by visual inspection, by stroboscopy, and by photon correlation spectroscopy; microcinematography and gradient centrifugation were occasionally used to clarify discrepancies. The motility of isolated spermatozoa could be maintained for periods up to 24 h at 4 or 37 degrees C; the presence of seminal fluid was not required and even provoked a reversible inhibition at 4 degrees C. Albumin facilitated cell movement between microscopic glass plates but had no effect on progressive motility per se, as evidenced by other techniques. During incubations of up to 2 h, progressive cell motility occurred independently of extracellular glucose and calcium but responded to variations in adenosine 3',5'-cyclic monophosphate and calcium. Dibutyryl cAMP increased forward motility, whereas ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid reversibly immobilized the spermatozoa in a calcium-dependent manner; phosphodiesterase inhibition resulted in increased vibration of sperm heads without any effect on progressive motility. Longer incubation periods required the presence of extracellular nutrients. These experiments further demonstrate that several motility measuring techniques should be used in parallel to distinguish the various components of cell movement, to exclude aspecific effects, and to supplement the shortcomings of each individual technique. Such procedure could clarify the various discrepancies that have been reported so far and should lead to a better understanding of the regulation of human sperm motility.

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