Unlocking sperm chromatin at fertilization requires a dedicated egg thioredoxin in Drosophila.
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ABSTRACT:
SUBMITTER: Tirmarche S
PROVIDER: S-EPMC5122968 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In Drosophila, male reproductive fitness can be affected by any number of processes, ranging from development of gametes, transfer to and storage of mature sperm within the female sperm storage organs, and utilization of sperm for fertilization. We have previously identified the 89B cytogenetic map position of D. melanogaster as a hub for genes that effect male paternity success when disturbed. Here, we used RNA interference to test 11 genes that are highly expressed in the testes and located within the 89B region for their role in sperm competition and male fecundity when their expression is perturbed. Testes-specific knockdown (KD) of bor and CSN5 resulted in complete sterility, whereas KD of CG31287, Manf and Mst89B, showed a breakdown in sperm competitive success when second to mate (P2 < 0.5) and reduced fecundity in single matings. The low fecundity of Manf KD is explained by a significant reduction in the amount of mature sperm produced. KD of Mst89B and CG31287 does not affect sperm production, sperm transfer into the female bursa or storage within 30 min after mating. Instead, a significant reduction of sperm in female storage is observed 24 h after mating. Egg hatchability 24 h after mating is also drastically reduced for females mated to Mst89B or CG31287 KD males, and this reduction parallels the decrease in fecundity. We show that normal germ-line expression of Mst89B and CG31287 is needed for effective sperm usage and egg fertilization.
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Project description:Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1-3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.
| S-EPMC5480393 | biostudies-literature
Project description:Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.
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Project description:Fertilization is an essential biological process in sexual reproduction and comprises a series of molecular interactions between the sperm and egg. The fusion of the haploid spermatozoon and oocyte is the culminating event in mammalian fertilization, enabling the creation of a new, genetically distinct diploid organism. The merger of two gametes is achieved through a two-step mechanism in which the sperm protein IZUMO1 on the equatorial segment of the acrosome-reacted sperm recognizes its receptor, JUNO, on the egg surface. This recognition is followed by the fusion of the two plasma membranes. IZUMO1 and JUNO proteins are indispensable for fertilization, as constitutive knockdown of either protein results in mice that are healthy but infertile. Despite their central importance in reproductive medicine, the molecular architectures of these proteins and the details of their functional roles in fertilization are not known. Here we present the crystal structures of human IZUMO1 and JUNO in unbound and bound conformations. The human IZUMO1 structure exhibits a distinct boomerang shape and provides structural insights into the IZUMO family of proteins. Human IZUMO1 forms a high-affinity complex with JUNO and undergoes a major conformational change within its N-terminal domain upon binding to the egg-surface receptor. Our results provide insights into the molecular basis of sperm-egg recognition, cross-species fertilization, and the barrier to polyspermy, thereby promising benefits for the rational development of non-hormonal contraceptives and fertility treatments for humans and other mammals.
| S-EPMC5319863 | biostudies-literature
Project description:Linker histone H1 has been studied in vivo and using reconstituted chromatin, but there have been few systematic studies of the effects of the cellular environment on its function. Due to the presence of many other chromatin factors and specific chaperones such as RanBP7/importin beta that regulate histone H1, linker histones likely function differently in vivo than in purified systems. We have directly compared H1 binding to sperm nuclei in buffer versus Xenopus egg extract cytoplasm, and monitored the effects of adding nuclear import chaperones. In buffer, RanBP7 decondenses sperm nuclei, while H1 binds tightly to the chromatin and rescues RanBP7-mediated decondensation. H1 binding is reduced in cytoplasm, and H1 exhibits rapid FRAP dynamics in cytoplasm but not in buffer. RanBP7 decreases H1 binding to chromatin in both buffer and extract but does not significantly affect H1 dynamics in either condition. Importin beta has a lesser effect than RanBP7 on sperm chromatin decondensation and H1 binding, while a combination of RanBP7/importin beta is no more effective than RanBP7 alone. In extracts supplemented with RanBP7, H1 localizes to chromosomal foci, which increase after DNA damage. Unlike somatic H1, the embryonic linker histone H1M binds equally well to chromatin in cytoplasm compared to buffer. Amino-globular and carboxyl terminal domains of H1M bind chromatin comparably to the full-length protein in buffer, but are inhibited ∼10-fold in cytoplasm. High levels of H1 or its truncations distort mitotic chromosomes and block their segregation during anaphase. RanBP7 can decondense sperm nuclei and decrease H1 binding, but the rapid dynamics of H1 on chromatin depend on other cytoplasmic factors. Cytoplasm greatly impairs the activity of individual H1 domains, and only the full-length protein can condense chromatin properly. Our findings begin to bridge the gap between purified and in vivo chromatin systems.
| S-EPMC2947519 | biostudies-literature
Project description:Fertilization is the culminating event in sexual reproduction and requires the recognition and fusion of the haploid sperm and egg to form a new diploid organism. Specificity in these recognition events is one reason why sperm and eggs from different species are not normally compatible. One notable exception is the unusual ability of zona-free eggs from the Syrian golden hamster (Mesocricetus auratus) to recognize and fuse with human sperm, a phenomenon that has been exploited to assess sperm quality in assisted fertility treatments. Following our recent finding that the interaction between the sperm and egg recognition receptors Izumo1 and Juno is essential for fertilization, we now demonstrate concordance between the ability of Izumo1 and Juno from different species to interact, and the ability of their isolated gametes to cross-fertilize each other in vitro. In particular, we show that Juno from the golden hamster can directly interact with human Izumo1. These data suggest that the interaction between Izumo1 and Juno plays an important role in cross-species gamete recognition, and may inform the development of improved prognostic tests that do not require the use of animals to guide the most appropriate fertility treatment for infertile couples.
| S-EPMC4275915 | biostudies-literature
Project description:In vivo doubled-haploid technology is widely applied in commercial maize breeding programs because of its time-saving and cost-reducing features. The production of maize haploids primarily depends on the use of Stock6-derived haploid inducer lines. Although the gene underlying haploid induction, MTL/ZmPLA1/NLD, was cloned recently, the mechanism of haploid induction is still unknown. Hetero-fertilization can occur via a single fertilization, which provides a means to investigate single-fertilization events by studying the hetero-fertilization phenomenon. In this study, we found that the hetero-fertilization rate increased significantly when female maize lines were first individually crossed with pollen from the inducer CAU5 in dual-pollination experiments 4 h before a second pollination with common lines. We also examined embryogenesis during haploid induction by confocal laser-scanning microscopy and observed single-fertilized ovules, indicating that single fertilization occurred during haploid induction. We therefore postulate that both single fertilization and chromosome elimination contribute to haploid induction in maize. We also propose a scheme for the formation of hetero-fertilized and haploid kernels. Our results provide an efficient approach to identify hetero-fertilized kernels for research on interactions between embryo and endosperm.
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Project description:Heterochromatin protein 1 (HP1) proteins are key factors of eukaryotic heterochromatin that coordinate chromatin compaction and transcriptional gene silencing. Through their multivalency they act as adaptors between histone H3 Lys9 di/trimethyl marks in chromatin and effector complexes that bind to the HP1 chromoshadow domain. Most organisms encode for multiple HP1 isoforms and the molecular mechanisms that underpin their diverse functions in genome regulation remain poorly understood. In fission yeast, the two HP1 proteins Chp2 and Swi6 assume distinct roles and Chp2 is tightly associated with the nucleosome remodeling and deacetylation complex SHREC. Here we show that Chp2 directly engages the SHREC nucleosome remodeler subunit Mit1. The crystal structure of the interaction interface reveals an extraordinarily extensive and specific interaction between the chromoshadow domain of Chp2 and the N terminus of Mit1. The integrity of this interface is critical for high affinity binding and for heterochromatin formation. Comparison with Swi6 shows that the Chp2-Mit1 interface is highly selective and thereby provides the molecular basis for the functional specialization of an HP1 isoform.
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Project description:Cysteine oxidation in protamines leads to their oligomerization and contributes to sperm chromatin compaction. Here we identify the Drosophila thioredoxin Deadhead (DHD) as the factor responsible for the reduction of intermolecular disulfide bonds in protamines and their eviction from sperm during fertilization. Protamine chaperone TAP/p32 dissociates DNA-protamine complexes in vitro only when protamine oligomers are first converted to monomers by DHD. dhd-null embryos cannot decondense sperm chromatin and terminate development after the first pronuclear division. Therefore, the thioredoxin DHD plays a critical role in early development to facilitate the switch from protamine-based sperm chromatin structures to the somatic nucleosomal chromatin.
| S-EPMC5238724 | biostudies-literature
Project description:During spermiogenesis in mammals and many other vertebrate classes, histone-containing nucleosomes are replaced by protamine toroids, which can repackage chromatin at a 10 to 20-fold higher density than in a typical somatic nucleus. However, recent evidence suggests that sperm of many species, including human and mouse retain a small compartment of nucleosomal chromatin, particularly near genes important for embryogenesis. As in mammals, spermiogenesis in the fruit fly, Drosophila melanogaster has also been shown to undergo a programmed substitution of nucleosomes with protamine-like proteins. Using chromatin immunoprecipitation (ChIP) and whole-genome tiling array hybridization (ChIP-chip), supported by immunocytochemical evidence, we show that in a manner analogous to nucleosomal chromatin retention in mammalian spermatozoa, distinct domains packaged by the canonical histones H2A, H2B, H3 and H4 are present in the fly sperm nucleus. We also find evidence for the retention of nucleosomes with specific histone H3 trimethylation marks characteristic of chromatin repression (H3K9me3, H3K27me3) and active transcription (H3K36me3). Raw and processed data from the experiments are available at GEO, accession GSE52165.
| S-EPMC4778661 | biostudies-literature