Protamine Replacement Research Articles

CDKN2AIP is critical for spermiogenesis and germ cell development

BackgroundAs a member of RNA-binding protein, CDKN2AIP has been shown to play a critical role in stem cell pluripotency and somatic differentiation. Recent studies indicate that Cdkn2aip is essential for spermatogonial self-renewal and proliferation through the activating Wnt-signaling pathway. However, the mechanisms of how Cdkn2aip regulate spermatogenesis is poorly characterized.ResultsWe discovered that the CDKN2AIP was expressed in spermatocyte as well as spermatids and participated in spermiogenesis. Cdkn2aip−/− mice exhibited multiple sperm head defects accompanied by age dependent germ cell loss that might be result of protamine replacement failure and impaired SUN1 expression. Loss of Cdkn2aip expression in male mice resulted in synapsis failure in 19% of all spermatocytes and increased apoptosis due to damaged DNA double-strand break (DSB) repair and crossover formation. In vitro, knockdown of Cdkn2aip was associated with extended S phase, increased DNA damage and apoptosis.ConclusionsOur findings not only identified the importance of CDKN2AIP in spermiogenesis and germ cell development, but also provided insight upon the driving mechanism.

PHF7 is a novel histone H2A E3 ligase prior to histone-to-protamine exchange during spermiogenesis.

Epigenetic regulation, including histone-to-protamine exchanges, controls spermiogenesis. However, the underlying mechanisms of this regulation are largely unknown. Here, we report that PHF7, a testis-specific PHD and RING finger domain-containing protein, is essential for histone-to-protamine exchange in mice. PHF7 is specifically expressed during spermiogenesis. PHF7 deletion results in male infertility due to aberrant histone retention and impaired protamine replacement in elongated spermatids. Mechanistically, PHF7 can simultaneously bind histone H2A and H3; its PHD domain, a histone code reader, can specifically bind H3K4me3/me2, and its RING domain, a histone writer, can ubiquitylate H2A. Thus, our study reveals that PHF7 is a novel E3 ligase that can specifically ubiquitylate H2A through binding H3K4me3/me2 prior to histone-to-protamine exchange.

Spermatid-specific linker histone HILS1 is a poor condenser of DNA and chromatin and preferentially associates with LINE-1 elements

BackgroundLinker histones establish and maintain higher-order chromatin structure. Eleven linker histone subtypes have been reported in mammals. HILS1 is a spermatid-specific linker histone, and its expression overlaps with the histone–protamine exchange process during mammalian spermiogenesis. However, the role of HILS1 in spermatid chromatin remodeling is largely unknown.ResultsIn this study, we demonstrate using circular dichroism spectroscopy that HILS1 is a poor condenser of DNA and chromatin compared to somatic linker histone H1d. Genome-wide occupancy study in elongating/condensing spermatids revealed the preferential binding of HILS1 to the LINE-1 (L1) elements within the intergenic and intronic regions of rat spermatid genome. We observed specific enrichment of the histone PTMs like H3K9me3, H4K20me3 and H4 acetylation marks (H4K5ac and H4K12ac) in the HILS1-bound chromatin complex, whereas H3K4me3 and H3K27me3 marks were absent.ConclusionsHILS1 possesses significantly lower α-helicity compared to other linker histones such as H1t and H1d. Interestingly, in contrast to the somatic histone variant H1d, HILS1 is a poor condenser of chromatin which demonstrate the idea that this particular linker histone variant may have distinct role in histone to protamine replacement. Based on HILS1 ChIP-seq analysis of elongating/condensing spermatids, we speculate that HILS1 may provide a platform for the structural transitions and forms the higher-order chromatin structures encompassing LINE-1 elements during spermiogenesis.

Pops gets the polycomb.

The paternal and maternal chromosomes of the early embryo are molecularly distinct at a key region of DNA that forms pericentric heterochromatin. It is there that the male DNA is bound by Polycomb Repressive Complex 1 (PRC1) proteins, and the female DNA is not. A new study in mice provides a molecular framework for this difference [1]. In mice, pericentric heterochromatin is built by 234-basepair-long AT-rich satellite repeat sequences. The exact function of these regions is still being investigated, but they may help to organize the nucleus and possibly attract other DNA domains for transcriptional silencing. The maternal pericentric heterochromatin contains H3K9me3 marks, which are known to foster DNA silencing. These marks are assembled on maternal DNA during oogenesis and persist through fertilization. In males, this mark is absent from pericentric heterochromatin—instead, PRC1 has taken over the silencing function. Mathieu Tardat et al. now outline, in molecular detail, how PRC1 is supplied to paternal DNA but is excluded from maternal DNA. A central player in this mechanism is Cbx2, a component of PRC1. It seems that one domain of Cbx2 fosters binding to AT-rich satellite regions, and a separate domain fosters binding to a histone mark specifically enriched on paternal pericentric heterochromatin H3K27me3. This mark is deposited onto paternal DNA sometime after the replacement of protamines by histones. Cbx2 is thwarted from binding to maternal DNA by Hp1b, a protein that binds to the histone marks enriched at maternal pericentric heterochromatin H3K9me3. Eliminating Hp1b in zygotes results in PRC1 binding to the maternal pericentric heterochromatin. The researchers say that this mechanism probably also operates in chromosomal regions outside of pericentric heterochromatin—PRC1, for instance, is found in regions of the paternal chromosomal arms in the early embryo. The differences between the maternal and paternal pericentric heterochromatin begin to disappear at the 8-cell stage, and the researchers speculate that this shift may be necessary to foster zygotic transcription and cell fate specification. The findings also showcase how different reprogramming histories during sperm and oocyte development influence the localization and function of chromatin-binding proteins at maternal and paternal DNA in the early embryo.

Identifying molecular and spermatological markers to detect sperm chromatin fragmentation

Transition nuclear proteins (TNPs) are the primary basic proteins produced from the histone replacement. Protamines (PRMs) and transition nuclear proteins are important for maintaining the sperm chromatin integrity, resistance to cryopreservational damage, potentiating fertilizing ability of sperm, preventing embryo deaths and abortions. Hence this histone to protamine replacement should be highly conserved among the males of different species. In the present study, blood and semen samples from 43 cattle and 17 buffalo bulls were collected to isolate genomic DNA and to study sperm chromatin fragmentation following varying period of cryopreservation. Single stranded conformational polymorphism (SSCP) was done to detect any single nucleotide polymorphisms (SNPs) in the coding exonic regions of TNP 1 and 2 as well as PRM 1 and 2 genes. The SSCP analysis did not reveal any SNP in these genes. This may be due to conserved pattern of these genes. The sperm chromatin fragmentation determined by comet assay was significantly different between the acceptable semen donating cattle as well as the buffalo bulls.

Dermatan sulfate synergizes with heparin in murine sperm chromatin decondensation

The mammalian sperm nucleus contains an unusually condensed chromatin, due to replacement of the majority of histones by protamines. However, soon after the spermatozoon penetrates the ooplasm at fertilization, decondensation of this densely packed chromatin must occur to allow formation of the male pronucleus and syngamy. Decondensation is accomplished by protamine disulfide bond reduction by oocyte glutathione and replacement of protamines by oocyte histones with the aid of an acceptor molecule. Previous results from our laboratory have demonstrated that heparan sulfate (HS) present in the ooplasm functions as protamine acceptor during human sperm decondensation in vivo. In the present paper, we analyze the role of heparin, structural analogue of HS, and dermatan sulfate (DS) in murine sperm chromatin decondensation in vitro, including the possibility of a synergistic effect between both glycosaminoglycans. Decondensation was assessed under phase contrast microscopy following incubation of murine spermatozoa with glutathione and either heparin, DS, or a combination of both. Ultrastructural changes taking place during decondensation were analyzed by transmission electron microscopy. Both glycosaminoglycans were able to promote the decondensation of murine spermatozoa in vitro but the decondensing ability of heparin was significantly higher. Use of both glycosaminoglycans together revealed the existence of a synergistic effect. Transmission electron microscopy analysis of decondensing spermatozoa supported these findings. Synergism between heparin and DS was observed both in capacitated and non-capacitated spermatozoa but decondensation kinetics was faster in the former. The results obtained indicate a new potential role for dermatan sulfate in murine sperm decondensation at fertilization and provide evidence of differences in the degree of chromatin condensation throughout the murine sperm nucleus.

Analysis of the methylation pattern of six gene promoters in sperm of men with abnormal protamination

It has recently been shown that alteration of the methylation pattern of imprinted genes is associated with different types of male infertility. The objective of our study was to investigate the methylation pattern of selected gene promoters in sperm of patients with abnormal protamine replacement. The promoters of OCT4, SOX2, NANOG, HOXC11, miR-17 and CREM were analyzed using bisulfite sequencing and the percentage of DNA methylation was compared between patients with an abnormal protamine 1/protamine 2 (P1/P2) ratio and normozoospermic controls. No significant quantitative differences were found between groups of patients with either an abnormally high or low P1/P2 ratio compared to normal controls. However, two individual samples from infertile subjects (2/20, 10%) showed an altered methylation pattern for the CREM gene promoter that was not found in control samples. These two samples had a significantly higher (P<0.05) promoter methylation (5.58 and 4.23%, respectively) compared to the control group (0.46%). In conclusion, in our pilot study, extreme methylations defects were not seen broadly in severely infertile men. However, two patients exhibited altered methylation of the CREM gene, which may be either causative or a result of abnormal protmaine replacement.

DNA methylation reprogramming and DNA repair in the mouse zygote

Here, we summarize current knowledge about epigenetic reprogramming during mammalian preimplantation development, as well as the potential mechanisms driving these processes. We will particularly focus on changes taking place in the zygote, where the paternally derived DNA and chromatin undergo the most striking alterations, such as replacement of protamines by histones, histone modifications and active DNA demethylation. The putative mechanisms of active paternal DNA demethylation have been studied for over a decade, accumulating a lot of circumstantial evidence for enzymatic activities provided by the oocyte, protection of the maternal genome against such activities and possible involvement of DNA repair. We will discuss the various facets of dynamic epigenetic changes related to DNA methylation with an emphasis on the putative involvement of DNA repair in DNA demethylation.

The aetiology of sperm protamine abnormalities and their potential impact on the sperm epigenome

During the elongating spermatid stage of spermatogenesis, there is a step-wise replacement of nuclear histones with protamines 1 and 2. In fertile men, the ratio of protamine 1/protamine 2 (P1/P2) is within the narrow range of 0.8-1.2. Ratios above or below that range are associated with infertility, exhibiting a wide range of defects including decreased sperm counts, morphology, fertilization ability, and embryo implantation capacity. In this review, we highlight studies evaluating potential causes of abnormal protamine expression, including the sequencing of genes relevant to protamine expression in both affected patients and controls. While the variants of the protamine genes themselves do not appear to be responsible for most observed defects, variants of the Contrin gene, a transcription factor and translation repressor, appear to be contributory to some cases of abnormal expression. Additionally, we explore the potential effects of abnormal protamine replacement on the epigenome of human sperm. Ongoing studies are evaluating the role of retained histones and DNA methylation in sperm, which may be affected in sperm with aberrant protamine replacement. This important area of epigenetic research has profound clinical implications.

The Drosophila Calcipressin Sarah Is Required for Several Aspects of Egg Activation

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.

217. Interaction of cdyl (chromodomain y-chromosome like) with the nuclear transport protein importin α2

Spermatogenesis is a unique, ordered process governed by the precise expression of a specific set of genes at each stage. Progression through successive stages requires the shuttling of proteins and transcription factors into and out of the nucleus to implement changes in gene transcription. Major factors that mediate nucleocytoplasmic transport are members of the importin superfamily, of which there are five and 20 different importin α and β genes, respectively, in mouse. We have previously demonstrated that several importins display distinct mRNA and protein expression patterns in adult mouse testis1 indicating that specific importins carry a specific cargo at discrete stages of spermatogenesis. Identification of importin cargoes in the testis should help describe the potential developmental switches critical to the spermatogenic process. We performed a yeast two-hybrid screen using full length importin α2 as bait and an adult mouse testis library, identifying nine target proteins. Some of these proteins include nuclear components that may be important in eliciting changes in the nuclear structure during spermatogenesis, as well as those involved in cell cycle regulation, homologous chromosome pairing and recombination, transcriptional regulation and guanine nucleotide biosynthesis. One key candidate is CDYL, which has been implicated in male infertility. It is a chromodomain-containing protein that is predominantly expressed during spermiogenesis and has been previously described to participate in hyperacetylation of histone H4, which is believed to facilitate protamine replacement of histones during spermiogenesis. Verification of CDYL-importin α2 interaction was demonstrated using co-immunoprecipation and co-transfection, while immunohistochemical staining of testis sections indicated colocalisation in the same cell types (mainly elongating spermatids). Importantly, preliminary experiments indicated that increasing CDYL nuclear accumulation by over-expressing importin α2 can increase histone H4 acetylation. Our hypothesis is that importin α2 is central in nuclear targeting of CDYL to facilitate its hyperacetylation role during protamine-histone exchange. (1)Hogarth et al. (2005). Dev. Dynamics (submitted).

6ACETYLATION OF HISTONE H4 LYSINE-5 AND LYSINE-8 DURING DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

The oocyte is remarkable in its ability to remodel the parental genomes following fertilization and to reprogram somatic nuclei as in nuclear transfer. While significant research has been carried out on DNA methylation patterns in the early embryo, increased interest in histone acetylation is more recent. The objective of this study was to characterize the pattern of acetylation of histone H4 lysine-5 (H4L5) and lysine-8 (H4L8) in the early pre-implantation bovine embryo. Bovine embryos were produced as previously described (Fouladi Nashta AA et al., 1998 Biol. Rep. 59, 255–262) and collected at different developmental stages, 1-cell (20h), 2-cell (30h), 4- and 8-cell (Day 2), 16-cell (Day 4), and blastocyst (Days 7–8) with an average of 6 embryos per group in two replicates. Embryos were fixed in 2.5% paraformaldehyde, 15min at room temperature (RT), stained with polyclonal rabbit antibodies against H4L5 (1:800) and H4L8 (1:600) residues (Serotec, UK) at 4°C overnight. A polyclonal swine anti-rabbit (1:200; Dako, Denmark) was used as secondary antibody for 40min at RT. Images were examined using a fluorescent microscope (Leica DMR, Germany). Image analysis and quantification were performed using Simple PCI software (Compix Imaging Systems, USA). Changes in intensities within and between different embryo stages were recorded as a ratio of red stain to blue counterstain. Data were corrected for confounding area and absorbance and analysed using a multivariate linear regression model. The intensity of staining for H4L5 appeared higher in 8-cell embryos than 2- and 4-cell embryos but not to a significant level (P≥0.05); 8-cell embryos also appeared higher in stain intensity than 16-cell but of borderline significance (P=0.073). Staining intensity decreased between the 8-cell and blastocyst stage (P≤0.05). In contrast, the intensity of acetylation staining for H4L8 residue decreased slightly between the 1- and 4-cell stages and then decreased significantly between the 4- and 8-cell stages (P≤0.05), increasing significantly by the 16-cell stage (P≤0.05). A significant decrease in staining intensity was observed at the blastocyst stage (P≤0.05). In blastocyst-stage embryos both lysine-5 and lysine-8 showed a differential staining of inner cell mass (ICM) and trophectoderm (TE) cells. ICM cells showed intense staining and TE cells stained very weakly. The intensity results presented are cumulative of ICM and TE intensities, which explains the overall low levels of acetylation in blastocysts when compared to the earlier stages. Acetylation of H4L5 starts high in 1-cell embryo, as it is necessary for protamine replacement (Adenot et al., 1997 Development 124, 4615–4625), decreases when methylation is high and increases when methylation is low (as in the 8-cell stage which corresponds with zygotic gene activation). Acetylation of H4L8 decreases between the 1-and 8-cell stages; however, its association with changes in DNA methylation has yet to be determined.

Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status

Objective To test, both in semen and washed-sperm fractions, whether hyaluronic acid (HA) binding is restricted to sperm that have completed cellular maturation. Design Comparisons of sperm in semen and in HA-bound sperm fractions. Setting University-based diagnostic and research andrology laboratory. Patient(s) Semen samples originated in men being tested for infertility. Intervention(s) The attributes of sperm maturity were tested by immunocytochemistry with creatine kinase and HspA2 antisera (highlights cytoplasmic retention in diminished-maturity sperm), aniline blue chromatin staining (detects persistent histones), pisum sativum lectin staining (reveals acrosomal integrity), and the FertiLight viability kit (highlights viable and nonviable sperm). Result(s) All markers of sperm maturity and immaturity supported the hypothesis that HA-bound sperm are mature. Nonbinding sperm exhibited cytoplasmic and nuclear properties of diminished maturity. The acrosomal status of HA-bound sperm was either unreacted or slightly capacitated, but not acrosome reacted. Only viable sperm exhibited HA binding. Conclusion(s) Sperm that are able to bind to HA are mature and have completed the spermiogenetic processes of sperm plasma membrane remodeling, cytoplasmic extrusion, and nuclear histone–protamine replacement. Hyaluronic acid–bound sperm show unreacted acrosomes. These studies provide further insights into the relationship between spermiogenesis and sperm function.

Effects of androgen-binding protein (ABP) on spermatid Tnp1 gene expression in vitro

In vitro studies were designed to determine whether Sertoli cell-delivered ABP could act on spermatogenetic events, whether such an action could occur via a paracrine or a juxtacrine pathway and whether sex steroids could be involved in this action. ABP delivery to germ cells was achieved using an in vitro model based on recombinant rat ABP-producing mouse Sertoli cells cocultivated with rat spermatids. Using semi-quantitative RT-PCR, the expression of the Tnp 1 gene encoding the Transition Protein 1, involved in the histone to protamine replacement during spermatid nuclear transformation, was analyzed. Our results provide clear evidence that Sertoli cell-derived ABP acts on spermatids by modifying the TP1 mRNA level. This outcome, strictly requiring juxtacrine conditions, is obtained in the absence of sex steroid hormones. To our knowledge this is the first evidence of an effect of ABP itself on male germ cells.

Hyperacetylation of histone H4 promotes chromatin decondensation prior to histone replacement by protamines during spermatogenesis in rainbow trout.

During the final stages of spermatogenesis in rainbow trout a dramatic increase in the level of histone H4 hyperacetylation is observed which is closely correlated with the replacement of histones by protamines. In order to understand further how H4 hyperacetylation might assist in protamine replacement of the histones, we have investigated the effect of H4 hyperacetylation on chromatin structure in trout testes actively undergoing the replacement process. Long chromatin fragments enriched in hyperacetylated H4 have been isolated and characterized. Evidence is presented that hyperacetylated H4 is clustered in certain regions (domains) of late stage testis chromatin and within these domains the chromatin exhibits an altered, highly relaxed structure which is believed to be the result of the extensive hyperacetylation. These domains, which are nearly devoid of protamine, are postulated to represent an initial structural transition which is necessary for the proper histone removal and protamine replacement process to take place.