Formation of native-like mammalian sperm cell chromatin with folded bull protamine

The DNA of most vertebrate sperm cells is packaged by protamines. The primary structure of mammalian protamine I can be divided into three domains, a central DNA binding domain that is arginine-rich and amino- and carboxyl-terminal domains that are rich in cysteine residues. In native bull sperm chromatin, intramolecular disulfide bonds hold the terminal domains of bull protamine folded back onto the central DNA binding domain, whereas intermolecular disulfide bonds between DNA-bound protamines help stabilize the chromatin of mature mammalian sperm cells. Folded bull protamine was used to condense DNA in vitro under various solution conditions. Using transmission electron microscopy and light scattering, we show that bull protamine forms particles with DNA that are morphologically similar to the subunits of native bull sperm chromatin. In addition, the stability provided by intermolecular disulfide bonds formed between bull protamine molecules within in vitro DNA condensates is comparable with that observed for native bull sperm chromatin. The importance of the bull protamine terminal domains in controlling the bull sperm chromatin morphology is indicated by our observation that DNA condensates formed under identical conditions with a fish protamine, which lacks cysteine-rich terminal domains, do not produce as uniform structures as bull protamine. A model is also presented for the bull protamine.DNA complex in native sperm cell chromatin that provides an explanation for the positions of the cysteine residues in bull protamine that form intermolecular disulfide bonds.     

Mapping and Measuring DNA to Protein Ratios in Mammalian Sperm Head by XANES Imaging

We have used XANES imaging, which combines X-ray absorption near edge spectral features (XANES) with 50-nm-resolution X-ray microscopy, to examine the content and distribution of DNA and protein in mature sperm cells. Sperm nuclei from five different species of mammals were examined; these species were chosen for analysis because their sperm contain marked differences in their protamine 1 and protamine 2 contents. The data we've obtained for bull, stallion, hamster, and mouse sperm suggest that the total nuclear protein to DNA ratio is similar in the sperm of many eutherian mammals. Since protamine constitutes the majority of the sperm nuclear protein, these results indicate that the total protamine content of sperm chromatin must be constant among mammalian species, independent of the extent of expression of the protamine 2 gene.     

Short communication: Extraction of sp18 family membrane proteins on posterior head of bull sperm and the effect of anti‐sp18 IgG on sperm motility and murine in vitro fertilization

Abstract

Bovine sperm heads were separated via ultrasonic treatment and centrifugation. Anti‐bull sperm IgG was produced by immunizing rabbits with acrosome‐reacted bull sperm heads. SDS PAGE patterns revealed that the main membrane proteins on acrosome‐reacted bull sperm head were sp18 family, including 18, 16, and 14 kD, which represented about 64% of the total membrane proteins in bull sperm. Indirect immunofluorescence shown sp18 antigens primarily distributed in postacrosomal and proximal tail regions. Western blot analysis revealed that the anti‐bull sperm IgG reacted with sp18 antigens in acrosome‐reacted bull sperm head and bull seminal plasma. Anti‐bull sperm IgG also reacted with 14, 16, 18, 42, 57 and 60 kD proteins in fresh bull, mouse and rabbit sperm. Anti‐sp18 IgG caused agglutination of bull and rabbit sperm, but had no effect on murine sperm. In murine in vitro fertilization trials, preincubating capacitated sperm with 0.364 mg/ml of anti‐sp18 IgG resulted in a decrease in the fertilization rate from 75.6% in the controls to 50.8% in the experimental groups (p<0.001).     

Zinc is sufficiently abundant within mammalian sperm nuclei to bind stoichiometrically with protamine 2

Although studies have demonstrated that zinc can bind to sperm nuclear proteins, specifically protamine 2, it has not been shown that the metal is sufficiently abundant inside the sperm nucleus to interact stoichiometrically with these proteins. In this study proton-induced X-ray emission (PIXE) has been used to measure the amount of sulfur and zinc within the nuclei of individual sperm cells to infer the stoichiometry of zinc binding to protamine 2 in six species of mammal: bull, chinchilla, stallion, hamster, human, and mouse (protamine 2 comprises from 0% (bull) to 67% (mouse) of the protamine present in the sperm of these animals). Using the sulfur mass and electrophoretic data on the relative proportion of protamine 1 and protamine 2 in the sperm chromatin of these species, the protamine 1, protamine 2, and total protamine contents within each species sperm nuclei have been determined. The PIXE measurements reveal that the zinc content of the sperm nucleus varies proportionately with the protamine 2 content of sperm chromatin. PIXE analyses of hamster protamines extracted under conditions that appear to at least partially preserve zinc binding also confirm that the majority of the metal is bound to protamine. In five of the species examined, sufficient zinc is present for each protamine 2 molecule to bind one zinc. The results obtained for chinchilla sperm, conversely, indicate the chinchilla protamine 2 molecule may interact differently with zinc. Chinchilla sperm only contain enough zinc for one atom to be bound to two protamine 2 molecules.     

Atomic force microscopy of mammalian sperm chromatin

We have used the atomic force microscope (AFM) to image the surfaces of intact bull, mouse and rat sperm chromatin and partially decondensed mouse sperm chromatin attached to coverglass. High resolution AFM imaging was performed in air and saline using uncoated, unfixed and unstained chromatin. Images of the surfaces of intact chromatin from all three species and of an AFM-dissected bull sperm nucleus have revealed that the DNA is organized into large nodular subunits, which vary in diameter between 50 and 100 nm. Other images of partially decondensed mouse sperm chromatin show that the nodules are arranged along thick fibers that loop out away from the nucleus upon decondensation. These fibers appear to stretch or unravel, generating narrow smooth fibers with thicknesses equivalent to a single DNA-protamine complex. High resolution AFM images of the nodular subunits suggest that they are discrete, clipsoid-shaped DNA packaging units possibly only one level of packaging above the protamine-DNA complex.     

Protamine-DNA association in mammalian spermatozoa

We have previously identified two subsets of basic nuclear proteins of mouse sperm: the protamines and a group of less basic proteins and, with the aid of a polyvalent antiserum, we have demonstrated their differential extractibility by NaCl in reducing solution (Rodman et al., J cell sci 53 (1982) 227) [9]. By affinity purification with isolated mouse sperm protamines we have obtained a protamine-specific fraction of that antiserum and a fraction that contains antibodies to the subset of less basic proteins. With those immunochemical probes we have shown the following The antigenic sites recognized by the protamine-specific antibodies are accessible, intranuclearly, only after the DNA has been removed by DNase I. The antibodies and DNA compete for binding sites on the protamines. DNA removal and consequent availability of the antigenic sites of the protamine molecules to the antibodies are possible only after displacement of the less basic proteins and chromatin decondensation have been induced. Immunoreactivity by the less basic proteins takes place without intervention of DNase. Those data indicate that the protamines are DNA-bound but that the less basic proteins are not or, alternatively, their putative DNA-binding sites do not coincide with their immuno-reactive sites. Those data also suggest that a function of the subset of less basic proteins may be to provide a shield for the protamine-DNA complex. The mouse protamine-affinity-bound antibodies are highly cross-reactive with protamines of other mammalian sperm suggesting that, despite considerable molecular diversity among mammalian protamines, the DNA-binding sites are conserved.     

Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry

To assess the structural stability of mammalian sperm nuclei and make interspecies comparisons, we microinjected sperm nuclei from six different species into hamster oocytes and monitored the occurrence of sperm nuclear decondensation and male pronucleus formation. The time course of sperm decondensation varied considerably by species: human and mouse sperm nuclei decondensed within 15 to 30 min of injection, and chinchilla and hamster sperm nuclei did so within 45 to 60 min, but bull and rat sperm nuclei remained intact over this same period of time. Male pronuclei formed in oocytes injected with human, mouse, chinchilla, and hamster sperm nuclei, but rarely in oocytes injected with bull or rat sperm nuclei. However, when bull sperm nuclei were pretreated with dithiothreitol (DTT) in vitro to reduce protamine disulfide bonds prior to microinjection, they subsequently decondensed and formed pronuclei in the hamster ooplasm. Condensed rat spermatid nuclei, which lack disulfide bonds, behaved similarly. The same six species of sperm nuclei were induced to undergo decondensation in vitro by treatment with DTT and detergent, and the resulting changes in nuclear size were monitored by phase-contrast microscopy and flow cytometry. As occurred in the oocyte, human sperm nuclei decondensed the fastest in vitro, followed shortly by chinchilla, mouse, and hamster and, after a lag, by rat and bull sperm nuclei. Thus species differences in sperm nuclear stability exist and appear to be related to the extent and/or efficiency of disulfide bonding in the sperm nuclei, a feature that may, in turn, be determined by the type(s) of sperm nuclear protamine(s) present.     

Formation of the perinuclear theca in spermatozoa of diverse mammalian species: relationship of the manchette and multiple band polypeptides

The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm: guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus.     

Sexual selection on protamine and transition nuclear protein expression in mouse species

Post-copulatory sexual selection in the form of sperm competition is known to influence the evolution of male reproductive proteins in mammals. The relationship between sperm competition and regulatory evolution, however, remains to be explored. Protamines and transition nuclear proteins are involved in the condensation of sperm chromatin and are expected to affect the shape of the sperm head. A hydrodynamically efficient head allows for fast swimming velocity and, therefore, more competitive sperm. Previous comparative studies in rodents have documented a significant association between the level of sperm competition (as measured by relative testes mass) and DNA sequence evolution in both the coding and promoter sequences of protamine 2. Here, we investigate the influence of sexual selection on protamine and transition nuclear protein mRNA expression in the testes of eight mouse species that differ widely in levels of sperm competition. We also examined the relationship between relative gene expression levels and sperm head shape, assessed using geometric morphometrics. We found that species with higher levels of sperm competition express less protamine 2 in relation to protamine 1 and transition nuclear proteins. Moreover, there was a significant association between relative protamine 2 expression and sperm head shape. Reduction in the relative abundance of protamine 2 may increase the competitive ability of sperm in mice, possibly by affecting sperm head shape. Changes in gene regulatory sequences thus seem to be the basis of the evolutionary response to sexual selection in these proteins.     

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.     

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     

Assessment of chromatin status (SCSA) in epididymal and ejaculated sperm in Iberian red deer, ram and domestic dog

Abnormal chromatin condensation is not detected using classical techniques for sperm analysis. SCSA has demonstrated its usefulness in sperm chromatin analysis in several species (human, bull, stallion and boar). In this work, we studied sperm samples from red deer, ram and dog to analyze the differentiation of chromatin structure applying SCSA in epididymal and ejaculated spermatozoa. Epididymal samples were obtained from the caput, corpus and cauda by means of cuts, and ejaculated ones were obtained by electroejaculation (deer), artificial vagina (ram) and digital manipulation (dog). SCSA results suggested different critical points in sperm maturation (spermatozoa with loose chromatin to more condensed chromatin) among species: from corpus to cauda in ram and from caput to corpus in deer and dog. Moreover, we also detected differences in ruminants and dog, reflected in the appearance of SCSA plots. Indeed, ram and deer samples rendered two peaks within the sperm main population (sperm with condensed chromatin), whereas only one was detected in dog. Although some differences were observed between cauda and ejaculated samples, SCSA parameters indicated good chromatin condensation, making these samples suitable for germplasm banking. Some species-dependent modifications in the analysis of the results may be necessary to take full advantage of its analytical power.     

Activation of mammalian oocytes by a factor obtained from rabbit sperm

In this study a fraction was prepared from rabbit sperm that activated rabbit and mouse oocytes following injection into the cytoplasm. The sperm factor activated oocytes exhibited cortical granule exocytosis, pronuclear formation, and cleavage. The sperm factor was soluble in aqueous solution and was not active extracellularly. Unlike most artificial activation methods that are only effective with aged oocytes, the sperm factor activated recently ovulated oocytes. The factor appears to be a protein or associated with a protein but not an acrosomal protein. Fractions from both mouse and bull sperm did not activate rabbit or mouse oocytes. Their inactivity may be owing to the techniques used to recover the fractions or differences between species in sperm morphology and fertilization processes. These observations support the hypothesis that oocyte activation is induced by a factor within sperm that is released into the cytoplasm of the oocyte at the time of sperm-oocyte fusion.     

Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.     

Dispersion of mammalian sperm chromatin during fertilization: an in vitro study

When "denuded spermatozoa" (spermatozoa stripped of the greater part of their acrosomes and resembling in may respects spermatozoa after acrosomal reaction) of the bull are incubated with 0.1 M 2-mercaptoethanol (pH 8), sperm chromatin is degraded extensively by a protease in the sperm head. The morphological pattern of sperm nuclear dispersion upon in vitro incubation is similar to that observed in the newly fertilized egg. Following disintegration of the outer layers of the sperm nucleus, chromatin dispersion commences from the periphery of the posterior half and proceeds to the anterior end and to the core of the head. Less basic N- and C-terminal portions of bull sperm histone molecules are digested quickly. The central, very arginine-rich portions of the molecules degrade gradually, yielding an heterogeneous series of arginine-rich peptides (molecular weight, 400-1500). Evidence suggests that the protease which is responsible for the degradation of sperm chromatin is a small fraction of acrosin. This fraction of acrosin appears to be arranged along the nuclear surface and to become associated with sperm chromatin during structural changes of the nuclear surface. A similar proteolysis of rabbit, hamster and guinea pig sperm chromatin has also been observed. The resulting pattern of dissolution of the sperm nucleus is proposed as a model of some of the steps involved in male pronucleus formation from the sperm head after fertilization. Histones H2a, H2b, H3, and H4 associated with DNA are relatively resistant to acrosin.     

Quantitative changes in histones during the formation of structures characteristic of the male pronucleus in mammalian spermatozoa in vitro

Treatment of bull spermatozoa with DDC--Na/dithiothreitol results in the swelling and decondensation of nuclear chromatin. The structures formed at the final stages of decondensation are morphologically similar to the male pronucleus. Cytophotometric analysis has shown that decondensation of chromatin in the gametes in followed by quantitative changes of basic nuclear proteins. In partly--decondensed sperm nuclei the intensity of histone staining increases as a result of the appearance of extra reactive groups. In fully decondensed nuclei there remain only 54% of histones of the original haploid level. Nucleoproteins revealed in the sperm with fully dispersed chromatin must be histones of the somatic type.     

Are fluorescent bodies of Y-spermatozoa detectable in common with mammalan species?

An attempt was made to detect the fluorescent bodies (F-body), using Quinacrine mustard (Q-M) staining in the spermatozoa from eight mammalian species (human, bull, boar, dog, rabbit, rat, mouse, and mastomys) as well as in the cock (used as negative control). Sperm suspension, prepared after rinsing by repeated centrifugation with phosphate buffered saline (PBS), was either stained with Q-M for 24 h or treated with protease and then stained with Q-M for 60 min. The final concentration of Q-M in the mixed staining sperm suspension was 0.025 mg/ml. The examination using a reflecting fluorescent microscope revealed that the F-body found in human sperm was also present in the sperm of all the mammals but not in the cock after 24 h of staining. The enzyme-treated specimens showed higher incidences of F-bodies than specimens stained for 24 h without enzymatic digestion. These findings strongly suggest that the F-body is commonly present in the spermatozoa of many mammalian species.     

Relationship between non-return rate and chromatin condensation of deep frozen bull spermatozoa

This study analyzes the relationship between chromatin condensation and field fertility, expressed as 90 days non-return rate (NRR), of bulls actively used by AI studs. Frozen-thawed semen from five bulls (six ejaculates per bull, three straws per ejaculate), that showed a non-return rate between 60 and 80%, were analyzed to assess sperm chromatin condensation and stability. The chromatin condensation was determined by flow cytometry using propidium iodide as fluorochrome, and the chromatin stability was evaluated by inducing its decondensation with SDS and EDTA. Coefficient of variation among replicates was less than 7% and 5% for chromatin condensation and stability, respectively. No correlation was present between chromatin condensation and NRR. However, significant correlation was found between chromatin stability and NRR. Chromatin stability was higher (P < 0.05) in those bulls that showed higher fertility. The results obtained in this study conclude that assessment of stability could be a valuable tool for routine evaluation and identification of ejaculates with high levels of sperm chromatin abnormalities and to detect animals of higher reproductive potential.