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Journal ArticleDOI

Drosophila protamine-like Mst35Ba and Mst35Bb are required for proper sperm nuclear morphology but are dispensable for male fertility.

01 Nov 2014-G3: Genes, Genomes, Genetics (Genetics Society of America)-Vol. 4, Iss: 11, pp 2241-2245

TL;DR: Drosophila males homozygous for a genomic deletion covering several genes including the protamine-like genes Mst35Ba/b are surprisingly fertile, and this work precisely deleted the Mst 35B locus by homologous recombination, and it is confirmed the dispensability of Mst34B for fertility.

AbstractDuring spermiogenesis, histones are massively replaced with protamines. A previous report showed that Drosophila males homozygous for a genomic deletion covering several genes including the protamine-like genes Mst35Ba/b are surprisingly fertile. Here, we have precisely deleted the Mst35B locus by homologous recombination, and we confirm the dispensability of Mst35Ba/b for fertility.

Topics: Drosophila Protein (56%), Protamine (54%), Homologous recombination (50%)

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Citations
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Journal ArticleDOI
TL;DR: The current knowledge of fertilization in Drosophila melanogaster is reviewed, with a special emphasis on the genes involved in the complex transformation of the fertilizing sperm nucleus into a replicated set of paternal chromosomes.
Abstract: The union of haploid gametes at fertilization initiates the formation of the diploid zygote in sexually reproducing animals. This founding event of embryogenesis includes several fascinating cellul...

38 citations


Journal ArticleDOI
27 Nov 2019-eLife
TL;DR: CidB targets nuclear-protein import and protamine-histone exchange and that CidA rescues embryos by restricting CidB access to its targets and this is proposed to be a rescue mechanism for embryos.
Abstract: Intracellular Wolbachia bacteria manipulate arthropod reproduction to promote their own inheritance. The most prevalent mechanism, cytoplasmic incompatibility (CI), traces to a Wolbachia deubiquitylase, CidB, and CidA. CidB has properties of a toxin, while CidA binds CidB and rescues embryonic viability. CidB is also toxic to yeast where we identified both host effects and high-copy suppressors of toxicity. The strongest suppressor was karyopherin-α, a nuclear-import receptor; this required nuclear localization-signal binding. A protein-interaction screen of Drosophila extracts using a substrate-trapping catalytic mutant, CidB*, also identified karyopherin-α; the P32 protamine-histone exchange factor bound as well. When CidB* bound CidA, these host protein interactions disappeared. These associations would place CidB at the zygotic male pronucleus where CI defects first manifest. Overexpression of karyopherin-α, P32, or CidA in female flies suppressed CI. We propose that CidB targets nuclear-protein import and protamine-histone exchange and that CidA rescues embryos by restricting CidB access to its targets.

37 citations


Cites background from "Drosophila protamine-like Mst35Ba a..."

  • ...PCR amplicons were produced with primers listed in Supplementary file 1i. High fidel- ity Phusion polymerase (New England Biolabs) was used to amplify DNA, which was then restriction enzyme digested, gel-purified and ligated into various plasmid vectors (Supplementary file 1j)....

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  • ...In order to precipitate and remove DNA, we added 5 M NaCl while stirring to a final concentration of 1 M and poly(ethyleneimine) (PEI) from a stock of 10% PEI in 10% HCl to a final concentration ~0.3–0.5%....

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  • ...The small, highly basic protamine proteins used to package paternal DNA at high density are stripped from the DNA, (Balhorn, 2007; Rathke et al., 2014; Tirmarche et al., 2014; Loppin et al., 2015; Tirmarche et al., 2016) and nucleosomes are then assembled with maternal histones (Loppin et al., 2015; Liu et al., 1997)....

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  • ...The small, highly basic protamine proteins used to package paternal DNA at high density are stripped from the DNA, (Balhorn, 2007; Rathke et al., 2014; Tirmarche et al., 2014; Loppin et al., 2015; Tirmarche et al., 2016) and nucleosomes are then assembled with maternal histones (Loppin et al....

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Journal ArticleDOI
TL;DR: It is demonstrated that the Drosophila maternal thioredoxin Deadhead (DHD) is specifically required to unlock sperm chromatin at fertilization and is then rapidly degraded after fertilization.
Abstract: In most animals, the extreme compaction of sperm DNA is achieved after the massive replacement of histones with sperm nuclear basic proteins (SNBPs), such as protamines. In some species, the ultracompact sperm chromatin is stabilized by a network of disulfide bonds connecting cysteine residues present in SNBPs. Studies in mammals have established that the reduction of these disulfide crosslinks at fertilization is required for sperm nuclear decondensation and the formation of the male pronucleus. Here, we show that the Drosophila maternal thioredoxin Deadhead (DHD) is specifically required to unlock sperm chromatin at fertilization. In dhd mutant eggs, the sperm nucleus fails to decondense and the replacement of SNBPs with maternally-provided histones is severely delayed, thus preventing the participation of paternal chromosomes in embryo development. We demonstrate that DHD localizes to the sperm nucleus to reduce its disulfide targets and is then rapidly degraded after fertilization.

33 citations


Journal ArticleDOI
20 Jul 2019-Gene
TL;DR: This review synthesize and summarize the current knowledge on the progress of chromatin remodeling during spermiogenesis, and straighten out the chronological order of chromatis remodeling and illustrate the possible regulation mechanisms of each step.
Abstract: The functional sperm is the key factor for species continuation. The process spermatogenesis, to produce mature sperm is quite complex. It begins with the proliferation and differentiation of spermatogonia, which develop from primary spermatocytes to secondary spermatocytes and round spermatids, which eventually develop into fertile mature sperm. Spermiogenesis is the latest stage of spermatogenesis, where the round spermatids undergo a series of dramatic morphological changes and extreme condensation of chromatin to construct mature sperm with species-specific shape. During spermiogenesis, chromatin remodeling is a unique progress. It leads the nucleosome from a histone-based structure to a mostly protamine-based configuration. The main events of chromatin remodeling are the replacement of histone by histone variants, hyperacetylation, transient DNA strand breaks and repair, variants by transition proteins and finally by protamines. In this review, we synthesize and summarize the current knowledge on the progress of chromatin remodeling during spermiogenesis. We straighten out the chronological order of chromatin remodeling and illustrate the possible regulation mechanisms of each step.

24 citations


Journal ArticleDOI
TL;DR: Data reveal that at least three chromatin-binding proteins act together in chromatin reorganization to compact the paternal chromatin, in agreement with independent loading of these factors into sperm chromatin.
Abstract: The formation of motile spermatozoa involves the highly conserved formation of protamine-rich, tightly packed chromatin. However, genetic loss of protamine function in Drosophila and mice does not lead to significant decompaction of sperm chromatin. This indicates that other proteins act redundantly or together with protamines. Here, we identify Prtl99C as a Drosophila sperm chromatin-associated protein that is essential for male fertility. Whereas the loss of protamines results in modest elongation of sperm nuclei, knockdown of Prtl99C has a much stronger effect on sperm nuclei. Loss of protamines and Prtl99C indicates an additive effect of these proteins on chromatin compaction, in agreement with independent loading of these factors into sperm chromatin. These data reveal that at least three chromatin-binding proteins act together in chromatin reorganization to compact the paternal chromatin.

12 citations


References
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Journal ArticleDOI
TL;DR: Comparison of protamine gene and amino-acid sequences suggests that the family evolved from specialized histones through protamine-like proteins to the true protamines.
Abstract: The protamines are a diverse family of small arginine-rich proteins that are synthesized in the late-stage spermatids of many animals and plants and bind to DNA, condensing the spermatid genome into a genetically inactive state. Vertebrates have from one to 15 protamine genes per haploid genome, which are clustered together on the same chromosome. Comparison of protamine gene and amino-acid sequences suggests that the family evolved from specialized histones through protamine-like proteins to the true protamines. Structural elements present in all true protamines are a series of arginine-rich DNA-anchoring domains (often containing a mixture of arginine and lysine residues in non-mammalian protamines) and multiple phosphorylation sites. The two protamines found in mammals, P1 and P2, are the most widely studied. P1 packages sperm DNA in all mammals, whereas protamine P2 is present only in the sperm of primates, many rodents and a subset of other placental mammals. P2, but not P1, is synthesized as a precursor that undergoes proteolytic processing after binding to DNA and also binds a zinc atom, the function of which is not known. P1 and P2 are phosphorylated soon after their synthesis, but after binding to DNA most of the phosphate groups are removed and cysteine residues are oxidized, forming disulfide bridges that link the protamines together. Both P1 and P2 have been shown to be required for normal sperm function in primates and many rodents.

540 citations


"Drosophila protamine-like Mst35Ba a..." refers background in this paper

  • ...Volume 4 November 2014 | Function of Protamine-Like Proteins | 2243...

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  • ...In mammals, the bulk of sperm chromatin is organized with two small protamines (Protamine 1 and 2) highly enriched in arginine residues (Balhorn 2007)....

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Journal ArticleDOI
TL;DR: It is found that a decrease in the amount of either protamine disrupts nuclear formation, processing of protamine-2 and normal sperm function, and that haploinsufficiency caused by a mutation in one allele of Prm1 or Prm2 prevents genetic transmission of both mutant and wild-type alleles.
Abstract: Protamines are the major DNA-binding proteins in the nucleus of sperm in most vertebrates and package the DNA in a volume less than 5% of a somatic cell nucleus. Many mammals have one protamine, but a few species, including humans and mice, have two. Here we use gene targeting to determine if the second protamine provides redundancy to an essential process, or if both protamines are necessary. We disrupted the coding sequence of one allele of either Prm1 or Prm2 in embryonic stem (ES) cells derived from 129-strain mice, and injected them into blastocysts from C57BL/6-strain mice. Male chimeras produced 129-genotype sperm with disrupted Prm1 or Prm2 alleles, but failed to sire offspring carrying the 129 genome. We also found that a decrease in the amount of either protamine disrupts nuclear formation, processing of protamine-2 and normal sperm function. Our studies show that both protamines are essential and that haploinsufficiency caused by a mutation in one allele of Prm1 or Prm2 prevents genetic transmission of both mutant and wild-type alleles.

475 citations


Journal ArticleDOI
TL;DR: It is found that ends-out targeting can be approximately as efficient as ends-in targeting, and is likely to be generally useful for Drosophila gene targeting.
Abstract: Ends-in and ends-out refer to the two arrangements of donor DNA that can be used for gene targeting Both have been used for targeted mutagenesis, but require donors of differing design Ends-out targeting is more frequently used in mice and yeast because it gives a straightforward route to replace or delete a target locus Although ends-in targeting has been successful in Drosophila, an attempt at ends-out targeting failed To test whether ends-out targeting could be used in Drosophila, we applied two strategies for ends-out gene replacement at the endogenous yellow (y) locus in Drosophila First, a mutant allele was rescued by replacement with an 8-kb y+ DNA fragment at a rate of ≈1/800 gametes Second, a wild-type gene was disrupted by the insertion of a marker gene in exon 1 at a rate of ≈1/380 gametes The I-SceI endonuclease component alone is not sufficient for targeting: the FLP recombinase is also needed to generate the extrachromosomal donor When both components are used we find that ends-out targeting can be approximately as efficient as ends-in targeting, and is likely to be generally useful for Drosophila gene targeting

364 citations


"Drosophila protamine-like Mst35Ba a..." refers background or methods in this paper

  • ...Volume 4 November 2014 | Function of Protamine-Like Proteins | 2243...

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  • ...…the simultaneous deletion of these other genetic elements could potentially interfere with a detailed functional analysis of Mst35B genes, we generated a precise deletion of the Mst35B locus by homologous recombination using the “EndsOut” targeting technique (Gong and Golic 2003, 2004) (Figure 1A)....

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Journal ArticleDOI
TL;DR: This review highlights the current knowledge on post-meiotic chromatin reorganization and reveals for the first time intriguing parallels in this process in Drosophila and mammals and illustrates the possible mechanisms that lead from a histone-based chromatin to a mainly protamine-based structure during spermatid differentiation.
Abstract: The function of sperm is to safely transport the haploid paternal genome to the egg containing the maternal genome. The subsequent fertilization leads to transmission of a new unique diploid genome to the next generation. Before the sperm can set out on its adventurous journey, remarkable arrangements need to be made during the post-meiotic stages of spermatogenesis. Haploid spermatids undergo extensive morphological changes, including a striking reorganization and compaction of their chromatin. Thereby, the nucleosomal, histone-based structure is nearly completely substituted by a protamine-based structure. This replacement is likely facilitated by incorporation of histone variants, post-translational histone modifications, chromatin-remodeling complexes, as well as transient DNA strand breaks. The consequences of mutations have revealed that a protamine-based chromatin is essential for fertility in mice but not in Drosophila. Nevertheless, loss of protamines in Drosophila increases the sensitivity to X-rays and thus supports the hypothesis that protamines are necessary to protect the paternal genome. Pharmaceutical approaches have provided the first mechanistic insights and have shown that hyperacetylation of histones just before their displacement is vital for progress in chromatin reorganization but is clearly not the sole inducer. In this review, we highlight the current knowledge on post-meiotic chromatin reorganization and reveal for the first time intriguing parallels in this process in Drosophila and mammals. We conclude with a model that illustrates the possible mechanisms that lead from a histone-based chromatin to a mainly protamine-based structure during spermatid differentiation. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.

342 citations


"Drosophila protamine-like Mst35Ba a..." refers background in this paper

  • ...Interestingly, the D. melanogaster genome contains several recent copies of Mst77F on the Y chromosome, and eight of these Mst77Y genes are most likely functional (Russell and Kaiser 1993; Krsticevic et al. 2010)....

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  • ...Russell, S. R., and K. Kaiser, 1993 Drosophila melanogaster male germ linespecific transcripts with autosomal and Y-linked genes....

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  • ...Drosophila comprises at least three SNBPs: two paralogous protamine-like proteins, Mst35Ba and Mst35Bb, which are conserved among drosophilids, and the HILS1related protein Mst77F (Russell and Kaiser 1993; Jayaramaiah Raja and Renkawitz-Pohl 2005; Alvi et al. 2013; Rathke et al. 2014)....

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Journal ArticleDOI
TL;DR: The most recent research into mammalian spermatozoal chromatin composition is discussed, supporting the hypothesis that the spermatozoon delivers a novel epigenetic signature to the egg that may be crucial for normal development and some thoughts on why this signature may be required in early embryogenesis.
Abstract: Haploid male germ cells package their DNA into a volume that is typically 10% or less that of a somatic cell nucleus. To achieve this remarkable level of compaction, spermatozoa replace most of their histones with smaller, highly basic arginine and (in eutherians) cysteine rich protamines. One reason for such a high level of compaction is that it may help optimise nuclear shape and hence support the gametes’ swimming ability for the long journey across the female reproductive tract to the oocyte. Super-compaction of the genome may confer additional protection from the effects of genotoxic factors. However, many species including the human retain a fraction of their chromatin in the more relaxed nucleosomal configuration that appears to run counter to the ergonomic, toroidal and repackaging of sperm DNA. Recent research suggests that the composition of this ‘residual’ nucleosomal compartment, a generally overlooked feature of the male gamete, is far more significant and important than previously thought. In this respect, the transport and incorporation of modified paternal histones by the spermatozoon to the zygote has been demonstrated and indicates another potential paternal effect in the epigenetic reprogramming of the zygote following fertilisation that is independent of imprinting status. In this review, the most recent research into mammalian spermatozoal chromatin composition is discussed alongside evidence for conserved, non-randomly located nucleosomal domains in spermatozoal nuclei, all supporting the hypothesis that the spermatozoon delivers a novel epigenetic signature to the egg that may be crucial for normal development. We also provide some thoughts on why this signature may be required in early embryogenesis. Reproduction (2010) 139 287–301

341 citations