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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.

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.
Abstract: During 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.

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Citations
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Journal ArticleDOI
TL;DR: It is shown that specifically reducing protamines induces distortion in an SD background, verifying that protamines are transported via the RanGAP/GEF pathway and indicating that E(SD) plays a significant and unique role in the process of distortion.
Abstract: Segregation Distorter (SD) is an autosomal meiotic drive system found worldwide in natural populations of Drosophila melanogaster. This gene complex induces the preferential and nearly exclusive transmission of the SD chromosome in SD/SD+ males. This selfish propagation occurs through the interplay of the Sd locus, its enhancers and the Rsps locus during spermatid development. The key distorter locus, Sd, encodes a truncated but enzymatically active RanGAP (RanGTPase-activating protein), a key nuclear transport factor in the Ran signaling pathway. When encoded by Sd, RanGAP is mislocalized to the nucleus interior, which then traps Ran inside the nucleus and disrupts nuclear import. As a result of this aberrant nuclear transport, a process known as the histone-to-protamine transition that is required for proper spermatid condensation fails to occur in SD/SD+ males. In this process, sperm-specific protamine proteins enter the spermatid nucleus and replace the formerly chromatin-complexed histones. Previously, we have shown that mutations affecting nuclear import and export can enhance distortion in an SD background, thus verifying that a defect in nuclear transport is responsible for the unequal transmission of chromosomes. Herein, we show that specifically reducing protamines induces distortion in an SD background, verifying that protamines are transported via the RanGAP/GEF pathway and indicating that E(SD) plays a significant and unique role in the process of distortion.

12 citations

Journal ArticleDOI
TL;DR: It is shown that repeated mating reduced the sperm pool and increased the percentage of GSCs in M- and S-phase of the cell cycle, and Germline expression of RNA-Interference constructs against G-proteins, or a dominant negative G-protein eliminated the increase in GSC division frequency in mated males.
Abstract: Adult stem cells divide to renew the stem cell pool and replenish specialized cells that are lost due to death or usage. However, little is known about the mechanisms regulating how stem cells adjust to a demand for specialized cells. A failure of the stem cells to respond to this demand can have serious consequences, such as tissue loss, or prolonged recovery post injury. Here, we challenge the male germline stem cells (GSCs) of Drosophila melanogaster for the production of specialized cells, sperm cells, using mating experiments. We show that repeated mating reduced the sperm pool and increased the percentage of GSCs in M- and S-phase of the cell cycle. The increase in dividing GSCs depended on the activity of the highly conserved G-proteins. Germline expression of RNA-Interference (RNA-i) constructs against G-proteins, or a dominant negative G-protein eliminated the increase in GSC division frequency in mated males. Consistent with a role for the G-proteins in regulating GSC division frequency, RNA-i against seven out of 35 G-protein coupled receptors (GPCRs) within the germline cells also eliminated the capability of males to increase the numbers of dividing GSCs in response to mating.

7 citations

Journal ArticleDOI
01 Sep 2015
TL;DR: It is suggested that the Drosophila Tpl94D orthologs should be classified as their own transition protein group based on the rigorous bioinformatic approach and the conservation of the HMG box reported in this work.
Abstract: The current study was aimed at analyzing putative protein sequences of the transition protein-like proteins in 12 Drosophila species based on the reference sequences of transition protein-like protein (Tpl (94D) ) expressed in Drosophila melanogaster sperm nuclei. Transition proteins aid in transforming chromatin from a histone-based nucleosome structure to a protamine-based structure during spermiogenesis - the post-meiotic stage of spermatogenesis. Sequences were obtained from NCBI Ref-Seq database using NCBI ORF-Finder (PSI-BLAST). Sequence alignments and analysis of the amino acid content indicate that orthologs for Tpl (94D) are present in the melanogaster species subgroup (D. simulans, D. sechellia, D. erecta, and D. yakuba), D. ananassae, and D. pseudoobscura, but absent in D. persmilis, D. willistoni, D. mojavensis, D. virilis, and D. grimshawi. Transcriptome next generation sequence (RNA-Seq) data for testes and ovaries was used to conduct differential gene expression analysis for Tpl (94D) in D. melanogaster, D. simulans, D. yakuba, D. ananassae, and D. pseudoobscura. The identified Tpl (94D) orthologs show high expression in the testes as compared to the ovaries. Additionally, 2 isoforms of Tpl (94D) were detected in D. melanogaster with isoform A being much more highly expressed than isoform B. Functional analyses of the conserved region revealed that the same high mobility group (HMG) box/DNA binding region is conserved for both Drosophila Tpl (94D) and Drosophila protamine-like proteins (MST35Ba and MST35Bb). Based on the rigorous bioinformatic approach and the conservation of the HMG box reported in this work, we suggest that the Drosophila Tpl (94D) orthologs should be classified as their own transition protein group.

6 citations


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

  • ...In flies, nuclear transformation involves the exchange of somatic histones for SNBPs called protamine-like proteins.21,22 In D. melanogaster, the transition protein Tpl94D facilitates the exchange of histones for protamine-like proteins.4-6 It has also been well documented that mammalian transition proteins (TPs) are involved in binding DNA to facilitate the transition from nucleosome-based chromatin to protamine-based chromatin.3 The D. melanogaster protamine-like proteins are male specific transcripts MST35Ba and MST35Bb.1,2,4,13,23 The purpose of MST35Ba and MST35Bb appears to be to serve as the protector of the compacted DNA in the sperm nucleus against detrimental environmental factors such as X-rays.6 Furthermore, deletion of MST35Ba and MST35Bb does not significantly affect chromatin *Correspondence to: Angela V Klaus, Email: angela.klaus@shu.edu Submitted: 01/14/2016; Revised: 04/08/2016; Accepted: 04/09/2016 http://dx.doi.org/10.1080/21565562.2016.1178518 www.tandfonline.com e1178518-1Spermatogenesis Spermatogenesis 5:3, e1178518; September/October/November/December 2015; Copyright © Taylor & Francis Group, LLC REPORT condensation or fertility as it does in mammals when true protamines are deleted.1,2,24,25 Recent studies showed that during spermiogenesis both transition (Tpl94D) and histone H1 linker-like (male specific transcript - MST77F) proteins play a significant role in remodeling the sperm nucleus in D. melanogaster.4,6 During sperm nuclear remodeling, the ubiquitous chromatin insulator protein CTCF has been postulated to be involved in controlling the areas where chromatin can undergo histone modification.4 These histone modifications include H2A mono-ubiquitination and an increase in H4 acetylation, which cause the histones on the chromatin to be removed and degraded.4 Consequently, an opening within the chromatin allows Tpl94D to act as an intermediate for the transition from a histone bound nucleosome to a protamine bound structure.4 A key component of Tpl94D that allows for chromatin condensation to occur is the N terminal high mobility group (HMG) box.4 This HMG box is rich in arginine, which is a very basic amino acid with high affinity for binding DNA.4,5 Recently, we performed a detailed bioinformatic analysis of protamine-like proteins in 12 species of Drosophila (D. melanogaster....

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  • ...melanogaster protamine-like proteins are male specific transcripts MST35Ba and MST35Bb.(1,2,4,13,23) The purpose of MST35Ba and MST35Bb appears to be to serve as the protector of the compacted DNA in the sperm nucleus against detrimental environmental factors such as X-rays....

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  • ...These sperm-specific proteins include histone H1 linker-like proteins,(1,2) true protamines,(3) protaminelike proteins,(1,2) chromatin insulator proteins,(4) and transition proteins.(2,4-6) Histone H1 linker-like proteins, true protamines and protamine-like proteins appear to have evolved from histone H1 linker and are collectively referred as the “sperm nuclear basic proteins” (SNBPs)....

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  • ...condensation or fertility as it does in mammals when true protamines are deleted.(1,2,24,25) Recent studies showed that during spermiogenesis both transition (Tpl) and histone H1 linker-like (male specific transcript - MST77F) proteins play a significant role in remodeling the sperm nucleus in D....

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Posted ContentDOI
09 Jun 2022-bioRxiv
TL;DR: SNBP rapid evolution is driven by genetic conflicts between sex chromosomes during spermatogenesis in Drosophila species, and it is hypothesized that SNBP genes ancestrally encoded by autosomes suppress meiotic drive, whereas sex-chromosomal SNBP expansions directly participate in meioticDrive.
Abstract: Many animal species employ short, positively charged proteins, called sperm nuclear basic proteins (SNBPs) or protamines, for tighter packaging of genomes in sperm. SNBP repertoires differ dramatically across animal lineages and signatures of rapid evolution have been reported in mammals. Both sperm competition and meiotic drive between sex chromosomes have been proposed as causes of SNBP innovation. We used a phylogenomic approach to investigate SNBP diversification and its underlying causes in Drosophila species. We found unambiguous signatures of positive selection in most SNBP genes except for genes essential for male fertility in D. melanogaster. Unexpectedly, evolutionarily young SNBP genes are more likely to encode essential functions for fertility than ancient, conserved SNBP genes like CG30056, which we found is dispensable for male fertility despite universal retention in Drosophila species. We found 19 independent amplification events involving eight SNBP genes that occurred preferentially on sex chromosomes in 78 Drosophila species. Conversely, we found that otherwise-conserved SNBP genes were lost in the montium group of Drosophila species, coincident with an X-Y chromosomal fusion. Furthermore, SNBP genes that became linked to sex chromosomes via chromosomal fusions are prone to degenerate or relocate back to autosomes. We hypothesize that SNBP genes ancestrally encoded by autosomes suppress meiotic drive, whereas sex-chromosomal SNBP expansions directly participate in meiotic drive. X-Y fusions in the montium group render autosomal SNBPs dispensable by making X-versus-Y meiotic drive obsolete or costly. We conclude that SNBP rapid evolution is driven by genetic conflicts between sex chromosomes during spermatogenesis in Drosophila species.

4 citations

Journal ArticleDOI
10 Feb 2023-eLife
TL;DR: Chang et al. as discussed by the authors used a phylogenomic approach to investigate SNBP diversification in Drosophila species and found that evolutionarily young SNBP genes are more likely to be critical for fertility than ancient, conserved genes.
Abstract: Many animal species employ sperm nuclear basic proteins (SNBPs) or protamines to package sperm genomes tightly. SNBPs vary across animal lineages and evolve rapidly in mammals. We used a phylogenomic approach to investigate SNBP diversification in Drosophila species. We found that most SNBP genes in Drosophila melanogaster evolve under positive selection except for genes essential for male fertility. Unexpectedly, evolutionarily young SNBP genes are more likely to be critical for fertility than ancient, conserved SNBP genes. For example, CG30056 is dispensable for male fertility despite being one of three SNBP genes universally retained in Drosophila species. We found 19 independent SNBP gene amplification events that occurred preferentially on sex chromosomes. Conversely, the montium group of Drosophila species lost otherwise-conserved SNBP genes, coincident with an X-Y chromosomal fusion. Furthermore, SNBP genes that became linked to sex chromosomes via chromosomal fusions were more likely to degenerate or relocate back to autosomes. We hypothesize that autosomal SNBP genes suppress meiotic drive, whereas sex-chromosomal SNBP expansions lead to meiotic drive. X-Y fusions in the montium group render autosomal SNBPs dispensable by making X-versus-Y meiotic drive obsolete or costly. Thus, genetic conflicts between sex chromosomes may drive SNBP rapid evolution during spermatogenesis in Drosophila species.In sperm, DNA is packaged more tightly than in other cells thanks to small proteins called ‘sperm nuclear basic proteins’ (SNBPs), also called protamines in mammals. SNBPs are important for sperm to develop properly and correctly perform their role during fertilization. Although the evolution of SNBPs has been studied in mammals, these proteins have not been as thoroughly examined in invertebrates. Chang et al. took advantage of the availability of high-quality sequences for the genomes of 78 species of Drosophila flies to investigate the evolution of the genes that code for SNBPs in these flies. The results showed that, just like in mammals, in Drosophila the protein sequences of SNBPs evolve rapidly. However, unlike mammals, Chang et al. also found that Drosophila species frequently gained and lost genes coding for SNBPs. Interestingly, the ‘older’ genes (genes that appeared earlier in evolution) that code for SNBPs are not essential for reproduction in the fruit fly Drosophila melanogaster. This is an unexpected finding because older genes usually have essential roles for survival and reproduction, which require them to be passed on to the next generation and remain in the genome. In contrast, younger SNBP genes that had appeared more recently and were not shared between different species of Drosophila were often essential for fertility. These results, combined with other observations about where SNBP genes are located in the genome, led Chang et al. to hypothesize that SNBPs present in sex chromosomes act as ‘meiotic drivers’ while those on other chromosomes (known as autosomes) suppress meiotic drive. In other words, SNBP genes present in the sex chromosomes may be responsible for killing sister sperm cells that do not carry those genes, while SNBP genes that are not located on sex chromosomes may suppress this activity. This is of particular interest because it indicates that SNBPs are involved in genetic conflicts between the two sex chromosomes: sperm that carry SNBPs on the X chromosome may kill sperm with a Y chromosome, and vice versa. The results of Chang et al. shed light on the mysterious evolution of SNBPs in Drosophila flies. Although previous hypotheses regarding the rapid evolution of SNBPs evolution have focused on their role in genome packaging, this new analysis suggests that much of the evolutionary change is likely driven by genetic conflicts between sex chromosomes.

4 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.

612 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.

518 citations

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.

411 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

381 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

374 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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