Assessment of DNA damage in sperm after repeated non‐invasive sampling in zebrafish Danio rerio

Repeated non‐invasive sampling of zebrafish Danio rerio sperm was conducted, sperm counts were obtained and a method for measurement of DNA damage in sperm was developed and validated (single‐cell gel electrophoresis, comet, assay). DNA damage in sperm increased with concentration of hydrogen peroxide (H₂O₂, 0–200 µM), and in vitro exposure of sperm to 200 µM H₂O₂ produced 88·7 ± 3·9% tail DNA compared to unexposed controls [12 ± 0·7% tail DNA (mean ± s.e ., n = 3)]. Frequency of sperm sampling (sampled every 2, 4 or 7 days) did not affect DNA damage in sperm, but sperm counts decreased 57 and 22% for fish sampled every 2 or 4 days, respectively.     

Effects of different extenders on DNA integrity of boar spermatozoa following freezing-thawing

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100 mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing–thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P < 0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100 mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100 mM trehalose, but cryopreservation could increase the degree of DNA damage (P < 0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.     

Estimates of nuclear DNA content in bryophyte sperm cells: phylogenetic considerations

Nuclear DNA contents of developing sperm were estimated for 17 species of bryophytes by cytophotometry in squash preparations of antheridia after Feulgen staining. Genome sizes are in the lower end of the range for land plants. Two homwort C-values have the lowest recorded for bryophytes at 0.17 and 0.26 pg DNA per nucleus. In liverworts, C-values range from 0.49 pg in Blasia pusilla to 4.05 pg in Pellia epiphylla, while moss genome sizes are less variable, ranging from 0.38 pg in Takakia ceratophylla to 0.92 pg in Atrichum oerstedianum. DNA content is not correlated with chromosome number in these bryophytes, but sperm cell size and cellular complexity are directly related to C-value. Structural variations in the locomotory apparatus are viewed as evolutionary modifications associated with changes in genomic complexity, with a generalized increase in complexity of the motile assemblage accompanying increases in DNA content. Nuclear DNA values are not as variable in bryophytes as they are in pteridophytes and seed plants. We suggest that in plants producing biflagellated gametes, lower DNA contents afford a selective advantage. Comparisons with plants that produce multiflagellated or pollen-dispersed sperm indicate operation of a nucleotypic effect in archegoniates with biflagellated sperm. This effect may be on sperm cell functioning, which in turn influences reproductive success.     

Artificial insemination in cattle with DNA‐treated sperm

Abstract

We have investigated the use of sperm cells as vectors for transferring exogenous DNA into the genome of cattle by artificial insemination with DNA‐treated sperm. First we demonstrated the DNA‐binding ability of cattle sperm with radioactively labeled DNA. For artificial insemination ejaculated semen was washed and incubated with 1 μg DNA/10⁶ sperm for one hour at 37°C. Three hundred synchronized heifers were inseminated once with a dose of 40×10⁶ sperm. Forty‐five calves and 41 fetuses were obtained. Southern analysis revealed in one calf a signal after probing with the 1 kb Pst I fragment of pSV2‐cat.     

DNA integrity of Polyodon spathula cryopreserved sperm

Comet assay was used to detect DNA integrity of paddlefish (Polyodon spathula) sperm following cryopreservation. At the same time, sperm velocities prior to freezing and post‐thawing were also assessed by the computer‐assisted sperm analysis (CASA) system. Significant differences (P < 0.05) were detected in the degree of DNA damage in cryopreserved sperm using different extenders. According to osmolality of the extenders, DNA damages of Sb (20 mm Tris, 75 mm sucrose, 0.5 mm KCl, pH 8.5) sperm was the least, which showed that the percentage of tail DNA of Sb (17.87–35.28%) was lower than those of Sa (20 mm Tris, 50 mm sucrose, 0.5 mm KCl, pH 8.5) and Sc (20 mm Tris, 100 mm sucrose, 0.5 mm KCl, pH 8.5). Moreover, A and B class sperm cells provided most of the Sb sperm (>50%). However, in light of the concentration of methanol, DNA damages of M8 (8% methanol concentration) sperm were the least, including a lower percentage of the tail DNA (21.56–30.86%), and C and D class sperm cells (<30%), regardless of the osmolality of the extenders. In conclusion, when the dilution was 20 mm Tris, 75 mm sucrose, 0.5 mm KCl, pH 8.5 and the concentration of methanol was 8%, the extenders were the best for cryopreservation of paddlefish sperm. In addition, the results indicated that the extent of damage to sperm motility caused by freeze‐thawing (VCL, VSL) was correlated with DNA breakage (|r| > 0.8). This implied that cryopreservation could damage sperm DNA of paddlefish and affect the sperm velocities when the osmolality and the concentrations of the cryoprotectants of the extender were inappropriate.     

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

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. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Preliminary study of sperm chromatin characteristics of the brachyuran crab Maja brachydactyla. Histones and nucleosome-like structures in decapod crustacean sperm nuclei previously described without SNBPs

An interesting characteristic of decapod crustacean sperm nuclei is that they do not contain highly packaged chromatin. In the present study we re-examine the presence of DNA-interacting proteins in sperm nuclei of the brachyuran Maja brachydactyla. Although previous reports have indicated that, unlike the majority of sperm cells, DNA of decapod sperm is not organized by basic proteins, in this work we show that: (1) histones are present in sperm of M. brachydactyla; (2) histones are associated with sperm DNA; (3) histone H3 appears in lower proportions than the other core histones, while histone H2B appears in higher proportions; and (4) histone H3 in sperm nuclei is acetylated. This work complements a previous study of sperm histones of Cancer pagurus and supports the suggestion that decapod crustacean sperm chromatin deserves further attention.     

Flow sorting of X and Y chromosome-bearing mammalian sperm: Activation and pronuclear development of sorted bull,boar, and ram sperm microinjected into hamster oocytes

Flow cytometric techniques were used to measure relative DNA content of X and Y chromosome-bearing bull, boar, and ram sperm populations and to separate the two sex-determining populations. Neat semen was prepared for flow cytometric analysis by washing, light sonication, and staining with 9 μM Hoechst 33342. Computer analysis of the bimodal histograms showed mean X-Y DNA differences of 3.9, 3.7, and 4.2% for bull, boar, and ram, respectively. Flow cytometric reanalysis of sorted bull, boar, and ram sperm showed purities greater than 90%. Bull, boar, and ram sperm nuclei were microinjected into hamster oocytes. Microinjected sperm were either unsorted, sorted, unsorted plus dithio-threitol (DTT) exposure, or sorted plus DTT exposure. Following microinjection, eggs were incubated 3 hr, fixed, and stained. A total of 579 eggs was observed for sperm activation (decondensation or formation of a male pronucleus). A lower percentage of sorted than unsorted (3 vs. 23%) boar sperm was activated (P <.05). However, sorted and unsorted DTT-exposed boar sperm or sorted and unsorted bull or ram sperm, regardless of DTT treatment, did not differ significantly. Sorted sperm nuclei of both rams and bulls exhibited higher activation rates than sorted boar sperm (P <.05). Treatment of sperm with DTT increased the activation rate (P < .05) for sorted boar sperm but not for bull or ram sperm. These data represent the first separation of bull, boar, and ram X and Y chromosome-bearing sperm populations and the first evidence that sperm of domestic animals sorted on the basis of DNA by flow cytometric procedures have the ability to decondense and to form pronuclei upon injection into a hamster egg.     

Potential for biparental cytoplasmic inheritance in Jasminum officinale and Jasminum nudiflorum

 Mature Jasminum officinale and J. nudiflorum pollen grains were stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined by epifluorescence microscopy. The pollen grains were found to be trinucleate, and the sperm cells in both species contained a large number of epifluorescent spots that corresponded to cytoplasmic DNA aggregates (nucleoids). The nucleoids of J. nudiflorum were observed to be dimorphic under the epifluorescence microscope, indicating that the sperm cells might contain both plastid and mitochondrial DNA. The nucleoids of J. officinale presented a similar appearance when stained with DAPI, but electron microscopic examination of the sperm cells revealed that they contained both plastids and mitochondria. When analyzed by DNA immunogold electron microscopy, gold particles were detected on both plastids and mitochondria. These findings demonstrated the preservation of plastid and mitochondrial DNA in mature sperm cells and thus the potential for biparental cytoplasmic inheritance in J. officinale and J. nudiflorum. Received: 8 August 1997 / Revision accepted: 25 February 1998     

DNA methylation stability in fish spermatozoa upon external constraint: Impact of fish hormonal stimulation and sperm cryopreservation

Highly differentiated mature spermatozoa carry not only genetic but also epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark toward reproductive biotechnologies is a major issue in animal production. The present work explored the impact of hormonal induction of spermiation and sperm cryopreservation in two cyprinids, the goldfish (Carassius auratus) and the zebrafish (Danio rerio), using LUminometric Methylation Assay (LUMA). We showed that while goldfish hormonal treatment did increase sperm production, it did not alter global DNA methylation of spermatozoa. Different sperm samples repeatedly collected from the same males for 2 months also showed the same global DNA methylation level. Similarly, global DNA methylation was not affected after cryopreservation of goldfish spermatozoa with methanol, whereas less efficient cryoprotectants (dimethylsulfoxide and 1,2‐propanediol) decreased DNA methylation. In contrast, cryopreservation of zebrafish spermatozoa with methanol induced a slight, but significant, increase in global DNA methylation. In the less compact nuclei, that is, goldfish fin somatic cells, cryopreservation did not change global DNA methylation regardless of the choice of cryoprotectant. To conclude, global DNA methylation is a robust parameter with respect to biotechnologies such as hormonal induction of spermiation and sperm cryopreservation, but it can be altered when the best sperm manipulation conditions are not met.     

Spermatozoa production by triploid males in the New Zealand freshwater snail Potamopyrgus antipodarum

Asexual lineages derived from dioecious taxa are typically assumed to be all female. Even so, asexual females from a variety of animal taxa occasionally produce males. The existence of these males sets the stage for potential gene flow across asexual lineages as well as between sexual and asexual lineages. A recent study showed that asexual triploid female Potamopyrgus antipodarum, a New Zealand freshwater snail often used as a model to study sexual reproduction, occasionally produce triploid male offspring. Here, we show that these triploid male P. antipodarum (1) have testes that produce morphologically normal sperm, (2) make larger sperm cells that contain more nuclear DNA than the sperm produced by diploid sexual males, and (3) produce sperm that range in DNA content from haploid to diploid, and are often aneuploid. Analysis of meiotic chromosomes of triploid males showed that aberrant pairing during prophase I probably accounts for the high variation in DNA content among sperm. These results indicate that triploid male P. antipodarum produce sperm, but the extent to which these sperm are able to fertilize female ova remains unclear. Our results also suggest that the general assumption of sterility in triploid males should be more closely examined in other species in which such males are occasionally produced. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 227–234.     

Seasonal changes in sperm DNA fragmentation of Murciano-Granadina goats: The compelling case for dynamic assessment

Evidence is provided of seasonal changes in the rate of DNA fragmentation dynamics of sperm collected from goats Murciano-Granadina breed), in a Spanish Breeding Centre at 41°N latitude. Results show that the initial assessment of sperm DNA fragmentation (T0) was not sufficient to differentiate sperm DNA fragmentation of bucks collected at different times of the year (Kruskal–Wallis 2.399; P = 0.493). However, when thawed semen was subsequently incubated at 37 °C for periods of between 2 and 48 h, differences were recorded in the rate of sperm DNA fragmentation (r-SDF), that are indicative of seasonal changes in reproduction (Log Rank: Mantel–Cox; χ² = 158.6; DF = 3; P < 0.001). Goat semen collected and cryopreserved in the Spanish winter and spring resulted in a poor post-thaw sperm DNA longevity and recorded a r-SDF of 0.8 and 1.3 per hour, after 48 h of incubation respectively. The r-SDF subsequently decreased to 0.16 and 0.36 per hour during summer and autumn. It is therefore, recommended that semen samples from Murciano-Granadina goats housed above the 40°N latitude be collected and cryopreserved in the summer and early autumn. This would result in a more stable DNA molecule, which is likely to improve the reproductive success.     

Transformation of the Hprt gene with DNA from spermatogenic cells

DNA-mediated transformation of hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient cells was used to assess the state of the X chromosome Hprt gene in spermatogenic cells. It had been shown previously that DNA from the inactive X chromosome of somatic cells functions poorly or not at all in HPRT transformation, indicating that DNA modification is involved in somatic cell X chromosome inactivation (XCI). In contrast, DNA from mature sperm does function in HPRT transformation suggesting that DNA modification may not be the basis of XCI in mature sperm. In this paper, transformation of HPRT^– mouse and hamster cells has been performed to test the nature of XCI during earlier stages of spermatogenesis. DNA from these developing murine germ cells was shown to be capable of HPRT transformation, extending the observation that XCI in sperm does not appear to involve a DNA modification. We also show here that DNA from mature sperm of marsupials functions in HPRT transformation, a result consistent with a role for sperm XCI in the evolution of somatic X inactivation.     

Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells ofPelargonium zonale during pollen development

Summary In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system     

Changes in DNA topology during spermatogenesis

DNA topology in histone- and protamine-depleted nuclei (nucleoids) from somatic cells, sperm, and spermatogenic cells was studied to determine if the superhelical configuration of DNA looped domains is altered during spermatogenesis. The expansion and contraction of nucleoid DNA was measured with a fluorescence microscope following exposure of nucleoids to different concentrations of ethidium bromide (EB). Nucleoids from Xenopus laevis erythrocytes, primary spermatocytes, and round spermatids, and from Rana catesbeiana sperm all exhibited a biphasic change (condensed-relaxed-condensed) in size as a function of exposure to increasing concentrations (0.5–100 g/ml) of EB, indicating that they contain negatively supercoiled DNA. In contrast, DNA in sperm nucleoids from Xenopus laevis and Bufo fowleri was relaxed and expanded at low (0.5–6 g/ml) EB concentrations, but became gradually condensed as the EB concentration was increased (6–100 g/ml). Nucleoids prepared from all cell types retained the general shape of the nucleus regardless of the superhelical configuration of the nucleoid DNA. Sperm nucleoid DNA condensed by 100 g/ml EB was relaxed by exposure to UV light, DNase I, proteinase K, or 4 M urea, but not by RNase A or 10 mM dithiothreitol. These results demonstrate that the DNA in sperm nucleoids is constrained in domains of supercoiling by nonbasic nuclear proteins. Negatively supercoiled DNA is present in nucleoids from cells with a full complement of histones, including Rana sperm, but not in nucleoids from Xenopus and Bufo sperm in which histones are replaced by intermediate-type protamines. Histone replacement in these species, therefore, is accompanied by unfolding of nucleosomal DNA and active removal of the negative supercoils. Results presented also suggest an important role for the nonbasic nuclear proteins of sperm in the morphogenesis of the nucleus and the arrangement of DNA.     

Nuclear matrix involvement in sperm head structural organization

Summary— In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.     

Association of goat (<Emphasis Type="Italic">Capra hircus</Emphasis>) CD4 gene exon 6 polymorphisms with ability of sperm internalizing exogenous DNA

As one of the transport systems on the sperm plasma membrane, CD4 molecule plays a distinct role in the process of sperm/DNA interaction. This makes it possible to explain the mechanism of sperm-mediated gene transfer (SMGT), which at present is still a mystery in this area. In this study, seminal samples of 60 individuals from seven breed bucks were collected to detect the ability of sperm in internalizing exogenous DNA, and genomic DNA from 147 individual blood samples (including 60 bucks referred ahead) were extracted to test the polymorphisms of CD4 genes by using PCR-SSCP technique. Then the correlation between them was evaluated. The results showed that: (1) it was a novel finding that breed-dependence of exogenous DNA binding to goat spermatozoa. There was the most significant difference among the buck breeds of sperm in binding exogenous DNA (F_{(6, 53)} = 4.811, P = 0.001) and in internalizing them into nuclei (F_{(6, 53)} = 4.587, P = 0.001). The ability of Lezhi Black goat was the lowest (P < 0.01) among the seven breeds. (2) There was no significant correlation between the ability of sperm in internalizing exogenous DNA and each semen quality parameter (P > 0.05). (3) In particular, three single-nucleotide polymorphisms (SNP) were described and there was one SNP (G/A⁷⁰⁰) of CD4 gene that made G234R substitution in the amino acid sequence of CD4 molecule. Nanjiang Yellow goat and Lezhi Black goat had higher hereditary variation compared with other breeds. (4) CD4 polymorphisms were highly associated with the ability of sperm in internalizing exogenous DNA. The SNP of Caprine CD4 gene exon 6 might be an important molecular marker of the ability to internalize exogenous DNA into sperm.     

Interaction of DNA with sperm-specific histones of the H1 family

Interactions of DNA with sperm-specific histones of the H1 family of sea urchin Strongylocentrotus intermedius, sea star Aphelasterias japonica, and bivalve mollusc Chlamis islandicus were studied using circular dichroism and the DNA melting analysis. Under physiological conditions, the highest DNA compacting ability was found in the echinoderm sperm H1 protein, in which additional α-helical domains are present in their C-terminal sequence. The derivative melting curves have two peaks: the low-temperature peak corresponds to the melting of free DNA, whereas the DNA regions bound to the protein melt at higher temperature. The highest stabilizing ability is characteristic of complexes with the mollusc sperm H1 protein.     

Association of Foreign DNA with Sperm of Gilthead Seabream, Sparus aurata, After Sonication, Freezing, and Dimethyl Sulfoxide Treatments

The association of foreign DNA with gilthead seabream (Sparus aurata) sperm was enhanced relative to simple coincubation by sonication, freezing, dimethyl sulfoxide and polyethylene glycol treatments. Sonication yielded the strongest dot blot signals, equivalent to 250 to 380 foreign DNA copies per spermatozoa. We are unaware of previous reports attempting to associate DNA with ultrasound for fish or elsewhere. However, no or negligible foreign DNA was evident in 1- and 2-day-old fish larvae resulting from eggs fertilized with sonicated or frozen sperm. Received October 14, 1997; accepted October 2, 1998     

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.