DNA In Sperm Nuclei Research Articles

Elucidation of DNA Packaging and Mispackaging in Sperm Nuclei by X-Ray Scattering

DNA in vertebrate sperm nuclei is organized differently from any other cell. DNA is compacted in tightly packed (∼ 400 mg/ml) hexagonal arrays. Since DNA repair is absent in sperm, dense packaging of DNA is considered necessary to protect against damage by reactive oxidizing species. DNA damage correlates with infertility and likely contributes to miscarriages and birth defects. Our goal is to characterize protamine defects that result in DNA mispackaging. Small (∼30-50 amino acids), arginine rich (50-70%) peptides called protamines assemble DNA in sperm nuclei. Protamines will spontaneously condense DNA from dilute solution into close-packed, liquid crystalline arrays. Both sperm nuclei and reconstituted protamine-DNA arrays are sufficiently ordered that the interhelical spacing or packing density can be determined by x-ray scattering.Insufficient protamine to neutralize all DNA has been linked to excess damage. Salmon sperm nuclei can be fractionated on sucrose density gradients. The interhelical scattering maxima of the heavy and light fractions overlap indicating that all nuclei have a tightly packed fraction of DNA. The lighter fraction, however, shows a scattering profile that is skewed toward larger spacings. Part of the DNA is not as well packed as the majority. Adding excess protamine to the light fraction recovers the scattering profile of the heavy fraction.Protamines are initially serine phosphorylated when replacing histones on DNA. Incomplete dephosphorylation has also been linked to DNA damage. Herring protamines can be partially phosphorylated with protein kinase A. We find that even modest phosphorylation that reduces the protamine charge by only 5% will increase the water volume accessible to oxidizing species in reconstituted DNA-protamine assemblies by 50%.

Sperm Nuclear Basic Proteins of Tunicates and the Origin of Protamines

Sperm nuclear basic proteins (SNBPs) are the chromosomal proteins that are found associated with DNA in sperm nuclei at the end of spermiogenesis. These highly specialized proteins can be classified into three major types: histone type (H-type), protamine-like type (PL-type), and protamine type (P-type). A hypothesis from early studies on the characterization of SNBPs proposed a mechanism for the vertical evolution of these proteins that involved an H1 → PL → P transition. However, the processes and mechanisms involved in such a transition were not understood. In particular, it was not clear how a molecular transition from a lysine-rich protein precursor (H1 histone) to the arginine-rich protamines might have taken place. In deuterostomes, the presence of SNBPs of the H-type in echinoderms and of protamines in the higher phylogenetic groups of vertebrates had long been known. The initial work on the characterization of tunicate SNBPs attempted to define the types and range of SNBPs that characterize this phylogenetically intermediate group. It was found that tunicate SNBPs belong to the PL-type. In this work we discuss how the study of SNBPs in the tunicates has been key to providing support to the H1 → PL → P transition. Most significantly, it was in tunicates that a potential molecular mechanism to explain the lysine-to-arginine transition was first reported.

Localization of single-stranded DNA in human sperm nuclei

To localize foci of single-stranded (ss) DNA in sperm nuclei of fertile and infertile men. Prospective, observational study. University infertility clinic. Semen samples from 12 consecutive asthenoteratospermic men presenting for infertility evaluation and 5 consecutive fertile men presenting for vasectomy. Semen analysis, semen processing, and immunocytochemistry using an antibody targeting ssDNA. Sperm nuclear ssDNA immunostaining pattern in whole and processed semen samples. Immunocytochemistry (using ssDNA antibody) demonstrated one of two sperm nuclear staining patterns: [1] faint punctated staining, and [2] diffuse nuclear staining. Infertile men had a higher proportion of spermatozoa exhibiting diffuse ssDNA staining than did fertile men (52 +/- 19 vs. 14 +/- 13, respectively). The proportion of spermatozoa exhibiting diffuse ssDNA staining was significantly higher in whole compared with processed semen. Positive (DNAse-treated nuclei) and negative controls (S1 nuclease-treated nuclei) were obtained to validate the specificity of the antibody. Human sperm nuclei generally exhibit discrete (presumably peripheral) foci of ssDNA. The data also show that infertile men have a higher proportion of sperm nuclei with diffuse areas of ssDNA than do fertile men, and suggest that spermatozoa with diffuse nuclear staining are abnormal.

Cytological Analyses in Melandrium album: Genome Size, C.hromosome Size and Fluorescence in situ Hybridization.

Melandrium album, a dioecious plant, has two sex chromosomes (X and Y). The haploid genome size of M. album was determined to be 4700 Mbp based on the amount of DNA in sperm nuclei in pollen by fluorimetry using a video-intensified microscope photon counting system (VIMPCS) after propidium iodide and 4', 6-diamidino-2-phenylindole staining. This value is about 33 times as much as that of Arabidopsis thaliana. The DNA content of each mitotic chromosome was also determined by VIMPCS. The size of the Y chromosome was 860 Mbp, which was 1.4 times as much as that of the X chromosome. Moreover, we visualized biotin-labeled 18S-5.8S-26S rRNA gene (large rRNA gene) probe and 5S rRNA gene probe in mitotic chromosomes by fluorescence in situ hybridization. Both of the rRNA genes were located on four pairs of autosomes, respectively. The sex chromosomes exhibited neither large rRNA genes nor 5S rRNA genes.

Cytochemical evaluation of sperm and lymphocyte DNA content after treatment with 5N HCl

In situ as well as extra situm cytochemical methods were used to investigate why the observed Feulgen-DNA value of sperm versus lymphocyte cells is lower than expected. After treatment with 5 N HCl, in situ experiments involving the GCA reaction and the UV cytophotometry showed the loss of DNA in sperm nuclei to be 12% more than that in lymphocyte nuclei. Extra situm study of sperm and lymphocytes treated with 5 N HCl showed the phosphate and DABA contents of sperm to be 35% and 23%, respectively, less than those of lymphocytes. The data suggest that sperm chromatin is much more sensitive than somatic chromatin to HCl depolymerization during the Feulgen reaction, and this can tentatively be attributed to the protein complement of sperm chromatin.

Quantification of the X- and Y-chromosome-bearing spermatozoa of domestic animals by flow cytometry.

The relative content of DNA in spermatozoa presumed to be the X- and Y-chromosome-bearing gametes from bulls, boars, rams and rabbits and the amount of DNA in spermatozoa of cockerels was determined by flow cytometry. Differences in the relative content of DNA and proportions of the presumed X- and Y-sperm populations in cryopreserved semen from Holstein, Jersey, Angus, Hereford and Brahman bulls were also determined. Spermatozoa were washed by centrifugation using a series of dimethyl sulfoxide solutions made in isotonic sodium citrate, fixed in ethanol, treated with papain and dithioerythritol to loosen the chromatin structure and remove cellular organelles, and stained quantitatively for DNA with the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). Approximately 5000 stained sperm nuclei, which were nonviable due to the removal of other cellular organelles during the washing procedure, were measured for DNA in an epi-illumination flow cytometer. A single distinct peak for cockerel spermatozoa and two symmetrical, overlapping peaks for species with X- and Y-spermatozoa were seen. This and other evidence strongly supports the interpretation that the peaks represent the X- and Y-sperm populations. The content of DNA in sperm nuclei from cockerels, bulls, boars, rams and rabbits, as determined by fluorescence flow cytometry, corresponded to biochemical estimates of DNA per sperm cell. Analyses of the bimodal histograms by computer-fitting two Gaussian distributions to the data showed the means of the peaks differed by 3.9, 3.7, 4.1 and 3.9% for bulls, boars, rams and rabbits, respectively. In four replicate analyses of semen from 25 bulls representing 5 breeds, the average population of sperm nuclei in the Y-peaks ranged from 49.5 to 50.5% for all breeds. The X-Y peak differences did not vary within each breed, but were significantly different when the breeds were compared. Spermatozoa from Jersey bulls had larger X-Y peak differences (P less than 0.001) than spermatozoa from Holstein, Hereford, and Angus bulls; spermatozoa from Brahman bulls had smaller X-Y differences (P less than 0.004). It is suggested from the evidence obtained in these studies that flow cytometry can be used to assess the proportion of X- and Y-spermatozoa in semen of domestic animals and is thereby applicable to verification of the effectiveness of enrichment techniques for X- or Y-spermatozoa.

Nuclear and chromatin structure in rat spermatozoa

AbstractThe nuclei of mature mammalian spermatozoa contain a highly ordered, lamellar substructure, presumably constituting the nucleoprotein of the haploid chromosomal complement. With a view toward constructing a plausible model of chromatin packing in sperm, we have determined some of the quantitative parameters associated with these “nuclear lamellae” in rat spermatozoa. Epididymal sperm from white, Sprague‐Dawley rats were examined by conventional sectioning methods, freeze fracture of fixed and unfixed specimens, and by whole mount replica techniques. Fixation and glycerolation did not significantly alter nuclear structure as seen by freeze fracture. Numerical data obtained from cross fractures of sperm heads indicate that the number of lamellae are quite constant at 10.4 ± 1.8 and that the linear measure of the lamellae is 7.2 ± 2.3 μm per cross fracture. The total area of cross fracture, assuming an elliptical profile is 2.3 k 0.7 μm2 and the thickness of the lamellae is 18.2 ± 3.5 nm with a range of 13.5 to 25.5 nm. An estimate of the total surface area of the nuclear lamellae could be made from measurements of projected nuclear area (from replicas and sections) as 173 ± 15 μm2. From these data and the known amount of DNA in the rat sperm nucleus, a model can be proposed for the organization of the nucleoprotein in these lamellar sheets. It is suggested that the chromatin is arranged in a coiled‐coil configuration closely associated together in a side‐by‐side fashion and continuous in extent. Approximate calculations based on this simple model are within a factor of 2 or 3 of predicting the correct amount of DNA in the sperm nucleus.

Molecular structure of chromatin during sperm differentiation of the dogfish Scyliorhinus caniculus (L.).

The molecular structure of chromatin during dogfish spermiogenesis was examined by electron microscopy after the dispersion of nuclei at low ionic strength. In early and late stages of differentiation (round and elongating spermatids), chromatin is globular, although basic nuclear proteins are different from those present in somatic nuclei. Three protein fractions are complexed with DNA in sperm nuclei. These fractions appear at the end of differentiation (elongated spermatids), subsequently undergoing a modification of their solubilization properties; only one protein fraction remains acid-soluble. Dispersed chromatin from sperm nuclei again shows a beads-on-a-string configuration both in the presence of the three specific sperm proteins and when the acid soluble fraction is extracted. Variations of the mean diameter of chromatin subunits during spermiogenesis appear rather limited compared to extensive modifications of chromatin superstructures.