Generation of a transgenic medaka (Oryzias latipes) strain for visualization of nuclear dynamics in early developmental stages

In this study, we verified nuclear transport activity of an artificial nuclear localization signal (aNLS) in medaka fish (Oryzias latipes). We generated a transgenic medaka strain expresses the aNLS tagged enhanced green fluorescent protein (EGFP) driven by a medaka beta‐actin promoter. The aNLS‐EGFP was accumulated in the nuclei of somatic tissues and yolk nuclei of oocytes, but undetectable in the spermatozoa. The fluorescent signal was observed from immediately after fertilization by a maternal contribution. Furthermore, male and female pronuclei were visualized in fertilized eggs, and nuclear dynamics of pronuclear fusion and subsequent cleavage were captured by time‐lapse imaging. In contrast, SV40NLS exhibited no activity of nuclear transport in early embryos. In conclusion, the aNLS possesses a strong nuclear localization activity and is a useful probe for fluorescent observation of the pronuclei and nuclei in early developmental stage of medaka.     

Alteration of poly(ADP-ribose) metabolism affects murine sperm nuclear architecture by impairing pericentric heterochromatin condensation

The mammalian sperm nucleus is characterized by unique properties that are important for fertilization. Sperm DNA retains only small numbers of histones in distinct positions, and the majority of the genome is protamine associated, which allows for extreme condensation and protection of the genetic material. Furthermore, sperm nuclei display a highly ordered architecture that is characterized by a centrally located chromocenter comprising the pericentromeric chromosome regions and peripherally positioned telomeres. Establishment of this unique and well-conserved nuclear organization during spermiogenesis is not well understood. Utilizing fluorescence in situ hybridization (FISH), we show that a large fraction of the histone-associated sperm genome is repetitive in nature, while a smaller fraction is associated with unique DNA sequences. Coordinated activity of poly(ADP-ribose) (PAR) polymerase and topoisomerase II beta has been shown to facilitate DNA relaxation and histone to protamine transition during spermatid condensation, and altered PAR metabolism is associated with an increase in sperm histone content. Combining FISH with three-dimensional laser scanning microscopy technology, we further show that altered PAR metabolism by genetic or pharmacological intervention leads to a disturbance of the overall sperm nuclear architecture with a lower degree of organization and condensation of the chromocenters formed by chromosomal pericentromeric heterochromatin.     

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

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 conclude that the appearance of precursor (intermediate) molecules facilitated the development of complex patterns of condensation and, as a consequence, a great diversity of forms in the sperm cell nuclei     

Sperm nuclear decondensation in mammals: role of sperm-associated proteinase in vivo

Previous studies from this (Zirkin et al., '80) and other (Marushige and Marushige, '78) laboratories have shown that proteinase associated with mammalian sperm nuclei is involved in thiol-induced sperm nuclear decondensation and protamine degradation in vitro. The results of these in vitro studies suggested the exciting possibility that the sperm nucleus itself might contribute proteinase involved in its subsequent in vivo decondensation during fertilization. In the present study, microinjection methods were used to test this possibility directly. Control hamster sperm nuclei, which exhibited proteinase activity, decondensed when incubated in vitro with disulfide reducing agent. As expected, these nuclei also decondensed when microinjected into ovulated hamster oocytes and formed morphologically normal pronuclei. When the proteinase associated with isolated sperm nuclei was removed with 0.5 M salt or inhibited with nitrophenyl-p-guanidinobenzoate, the nuclei were rendered incapable of decondensing in response to disulfide reducing agent in vitro. However, when these nuclei were microinjected into eggs, they decondensed and transformed into pronuclei. These results provide direct evidence that sperm-associated proteinase is not required for sperm nuclear decondensation and formation of the male pronucleus during fertilization.     

Ability of hamster spermatozoa to digest their own DNA

Mammalian sperm chromatin is bound by protamines into highly condensed toroids with approximately 50 kilobases (kb) of DNA. It is also organized into loop domains of about the same size that are attached at their bases to the proteinaceous nuclear matrix. In this work, we test our model that each sperm DNA-loop domain is condensed into a single protamine toroid. Our model predicts that the protamine toroids are linked by chromatin that is more sensitive to nucleases than the DNA within the toroids. To test this model, we treated hamster sperm nuclei with DNase I and found that the sperm chromatin was digested into fragments with an average size of about 50 kb, by pulse-field gel electrophoresis (PFGE). Surprisingly, we also found that spermatozoa treated with 0.25% Triton X-100 (TX) and 20 mM MgCl2 overnight resulted in the same type of degradation, suggesting that sperm nuclei have a mechanism for digesting their own DNA at the bases of the loop domains. We extracted the nuclei with 2 M NaCl and 10 mM dithiothreitol (DTT) to make nuclear halos. Nuclear matrices prepared from DNase I-treated spermatozoa had no DNA attached, suggesting that DNase I digested the DNA at the bases of the loop domains. TX-treated spermatozoa still had their entire DNA associated with the nuclear matrix, even though the DNA was digested into 50-kb fragments as revealed by PFGE. The data support our donut-loop model for sperm chromatin structure and suggest a functional role for this type of organization in that sperm can digest its own DNA at the sites of attachment to the nuclear matrix.     

Involvement of an acrosinlike proteinase in the sulfhydryl-induced degradation of rabbit sperm nuclear protamine

Previous studies demonstrated that proteolytic activity is associated with isolated rabbit sperm nuclei and is responsible for the degradation of nuclear protamine that occurs during thiol-induced in vitro decondensation of the nuclei (Zirkin and Chang, 1977; Chang and Zirkin, 1978). In this study, we present the results of experiments designed to characterize this proteolytic activity. Basic protein isolated from rabbit sperm nuclei incubated with 5 mM dithiothreitol (DTT) and 1 percent Triton X-100 for increasing periods of time exhibited progressively faster migrating bands on acid-urea polyacrylamide gels, reflection the progressive degradation of protamine. Ultimately, a specific and characteristic peptide banding pattern resulted. When sperm nuclei were treated with the esterase inhibitor nitrophenyl-p-guanidino benzoate (NPGB) to inhibit the nuclear-associated proteolytic activity and then incubated with one of several exogenous proteinases in addition to DTT and Triton X-100, characteristic peptide banding patterns were seen for each exogenous proteinase employed. For trypsin, chymotrypsin, pronase, and papain, the peptide banding patterns differed from one another and from the pattern characteristic of protamine degradation by the nuclear-associated proteinase. By contrast, when rabbit acrosin served as the exogenous proteinase, the peptide banding pattern seen was identical to the pattern characteristic of the nuclear-associated proteinase. These results demonstrate directly that the proteinase associated with rabbit sperm nuclei and involved in sperm nuclear decondensation in vitro is acrosinlike.     

Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.     

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

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.     

Proteolytic degradation of protamine during thiol-induced nuclear decondensation in rabbit spermatozoa

Rabbit sperm nuclei decondensed when incubated with dithiothreitol and Triton X-100. Protamine isolated from these nuclei during the course of treatment exhibited marked and progressive degradation. Timed interval experiments revealed that the degradation of protamine significantly preceded sperm nuclear decondensation and reached its maximum extent before decondensation was completed. Studies with protease inhibitors demonstrated that proteolysis was involved in both nuclear decondensation and protamine degradation.     

Further evidence that sperm nuclear proteins are necessary for embryogenesis

We have recently presented evidence that the structural integrity of the mouse sperm nuclear matrix may be necessary for the proper unpackaging of sperm DNA for participation in embryogenesis. It is likely that the sperm nuclear matrix contributes to the organisation of the sperm DNA and its disturbance can seriously damage the paternal genome or its expression. In this work, we confirm our previous data and further suggest that even very subtle changes in the sperm nuclear structure may have a significant impact on embryo development. As reported previously, dithiothreitol (DTT) in the presence of an ionic detergent, ATAB, destabilized the nuclear matrix as measured by the halo assay, and oocytes injected with these nuclei failed to develop. We also discovered that omitting the protease inhibitor PMSF from the buffers used to extract spermatozoa prevented sperm injected into oocytes from participating in development. The organization of DNA into loop domains by the nuclear matrix in these nuclei appeared normal, as measured by the halo assay. Oocytes injected with sperm nuclei that had been washed with ATAB in the presence of phenylmethylsulphonyl fluoride (PMSF) but in the absence of DTT resulted in live births. Neither DTT treatment nor the absence of PMSF would be expected to disrupt the integrity of the paternal DNA. The data therefore suggest that even very subtle alterations in the structural proteins of the nucleus are enough to deprive sperm DNA of the ability to contribute to embryonic development.     

The lack of protamine 2 (P2) in boar and bull spermatozoa is due to mutations within the P2 gene.

The nuclei of spermatozoa in all mammals examined so far contain P1 protamine. A second protamine variant, protamine P2, has to date been isolated only from human and murine spermatozoa where it represents the major fraction of basic nuclear protein. In order to elucidate the reason for this unusual distribution of the protamine variants among mammals we have investigated the expression of protamine P2 in boar and bull. It can be shown that also in these species protamine 2 is transcribed and translated on low levels. Various mutational events though have altered the primary structure of the protein: In boar, a deletion of 8 aminoacids has removed a sequence motif from the amino-terminus of the molecule, which highly probable is of functional relevance. The bovine sequence, as a consequence of numerous point mutations has accumulated neutral and hydrophobic aminoacids which reduce the affinity of the protamine 2 to DNA.     

Spermiogenesis and chromatin condensation in the common tree shrew, <Emphasis Type="Italic">Tupaia glis</Emphasis>

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. This work was supported by the Thailand Research Fund (Senior Research Fellowship to Prof. Prasert Sobhon).     

Chromatin condensation in hamster sperm: A flow cytometric investigation

In this study we have used acridine orange staining, as described by Evenson (1990), to follow changes in DNA packaging as they occur in hamster spermatozoa which have left the testis and are undergoing maturation in the epididymis. Measurement of the green and red fluorescent intensities of hamster sperm nuclei by flow cytometry demonstrated a decrease in acridine orange binding to DNA as sperm made their way from proximal corpus epididymis to the vas deferens. Using sperm from the cauda epididymis of the mature hamster as the standard, a method was developed for estimating the % of cells in a given sample that have matured with regard to DNA packaging. Staining with bromobimane was used to determine the extent of sulfhydryl oxidation in the nuclei. It was seen that sulfhydryl oxidation occurred mainly in the cauda epididymis whereas another process in chromatin condensation occurred earlier, during sperm passage through the caput epididymis. This earlier process could be mimicked by incubating sperm nuclei with alkaline phosphatase, suggesting that it consists of removal of phosphate in protamine. © 1994 Wiley-Liss, Inc.     

Sperm nuclear transformations consist of enlargement and condensation coordinate with stages of meiotic maturation in fertilized Spisula solidissima oocytes

Rates of sperm nuclear expansion were measured and correlated with processing of the maternal chromatin in synchronous populations of fertilized surf clam (Spisula solidissima) oocytes fixed at regular intervals following insemination and stained with the DNA fluorochrome Hoechst 33342. Sperm nuclei expanded in four distinct phases each temporally coordinate with events of meiotic maturation: germinal vesicle stage (phase A), germinal vesicle breakdown (phase B), polar body formation (phase C), and female pronuclear development (phase D). Sperm nuclei were essentially unchanged during phase A (rate = 0.1 micron2/min, enlarged during phases B (rate = 8.2 microns2/min) and D (rate = 6.2 microns2/min), and condensed during phase C (rate = -1.9 micron2/min). Sperm nuclear enlargement during phase D was significantly less in polyspermic and polygynic zygotes. The effects of various treatments (temperature, microtubule disruption, pH alterations, and metabolic and protein synthesis inhibitions) which perturbed sperm nuclear enlargement and meiotic processing of the maternal chromatin indicated that the two processes are coupled and may be linked by common regulatory agents.     

Sperm nuclear halos can transform into normal chromosomes after injection into oocytes

Mouse sperm nuclei extracted with an ionic detergent and 2 M NaCl retain their overall morphology, but upon subsequent reduction of the protamine disulfides they lose all elements of chromatin structure except the organization of DNA into loop that are anchored to the nuclear matrix. These DNA loops appear as a halo surrounding the nuclear matrix, and nuclei extracted in this manner are, therefore, called nuclear halos. Here, we report that sperm nuclear halos injected into oocytes can form pronuclei, then transform into chromosomes with normal morphology. This suggests that sperm nuclear halos retain all the information necessary for normal chromosomal organization, and that micromanipulation of these extracted sperm nuclei can be accomplished without major DNA damage.     

Electrophoretic analysis of nuclear proteins isolated from sperm of the black abalone Haliotis crackeroidii

Nuclear proteins isolated from spermatozoa of the abalone, Haliotis crackeroidii, after sonication and CTAB treatment were characterized electrophoretically. Mature, sonication-resistant sperm nuclei contain only protamine and lack detectable levels of somatic histone. The amino acid composition and size of this protamine suggests that its structure must be intermediate between fish and mammalian protamines.     

Effect of sperm cryopreservation on sperm DNA stability and progeny development in rainbow trout

This study was carried out to test how sperm cryopreservation affected nuclear DNA stability and whether progeny development was modified when eggs were fertilized with cryopreserved spermatozoa. The "comet assay" (alkaline single-cell gel electrophoresis assay) was adapted to trout spermatozoa to estimate DNA stability as measured by alkali-induced DNA strand break formation. Because trout eggs develop in water after fertilization (oviparous species) and that eggshell is easy to clear up after fixative treatment, progeny development was assessed from the blastodisc flattening stage of the embryos to the first feeding stage of the hatched fries by direct observation. All parameters under study were analyzed on each sperm and comparisons between parameters were made using paired data. Freeze-thawing of sperm slightly but significantly increased the percentage of nuclei showing altered DNA after comet assay. This increase was correlated to the decrease in fertilization rates of sperm, but the absolute percentage of altered nuclei was not predictive of the absolute fertilization ability of sperm. Assessment of progeny development showed that survival rate and abnormality rate obtained after fertilization with cryopreserved sperm were not different from those obtained with fresh sperm. It is concluded that trout sperm cryopreservation only slightly affected sperm DNA stability and that the use of cryopreserved spermatozoa did not impair offspring survival and quality.     

Phosphorylation state of protamines 1 and 2 in human spermatids and spermatozoa

The basic nuclear proteins of a fraction of elongating spermatids from human tests and of a fraction of motile spermatozoa from the ejaculate, separated by ion-exchange chromatography, were compared. Analysis by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCI) while protamine 2 alone was eluted by 50% GuCI. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation-dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined. The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human spermatozoa.