Polymorphism detection and characterization of sperm cells chromatin remodeling associated genes in Murrah buffalo.

Seasonal variations significantly impact buffalo bull semen production and quality, particularly during the summer months. Understanding the genetic basis of these changes is important for managing bull fertility and improving sperm quality. The present study focused on characterizing and identifying polymorphisms in chromatin remodeling genes, protamines (PRMs) and Transition Nuclear Proteins (TNPs) in Murrah buffalo bulls with varying semen quality due to seasonal effects. Our findings revealed none of the coding region variation in PRM1, PRM2, TNP1, and TNP2, these genes are highly conserved in buffalo. Two intronic variants were identified, including G16C in PRM1 intron 1 and intronic SNP in PRM2 intron 1 (G96A). The complete CDS of consensus sequence of bubaline PRM1 was 86.3% identical and 94.1% similar to the bovine PRM1. Whereas the complete CDS of consensus sequence of bubaline TNP2 was 78.2% identical and 91.0% similar to bovine TNP2. Further, no statistically significant differences in the fold change of TNP1, TNP2, PRM1, and PRM2 levels between the hot summer SNA and SA groups and the winter SNA and SA groups This study represents the first comprehensive report on the characterization of bubaline PRM1 (complete CDS), PRM2 (partial CDS), TNP1 (partial CDS), and TNP2 (complete CDS) genes in buffalo sperm cells. Results of the study, clearly indicate that the genes associated with protamine (PRM1 and TNP2) are highly conserved in Bubalus bubalis. Understanding these genetic underpinnings can have implications for improving buffalo bull fertility and semen quality.

Monitoring Binding of Protamine with DNA Using Protein Charge Transfer Spectra.

In this work, novel intrinsic electronic absorption (250-400 nm) with a molar extinction coefficient of 752 M-1cm-1 at 250 nm, arising from photoinduced electron transfer involving charged amino acid side chains and the polypeptide backbone, along with luminescence (300-500 nm) with quantum yield of 0.011 from subsequent charge recombination, was observed in salmon sperm Protamine (PRM). The absorption of PRM was attributed to the previously identified Peptide Backbone-to-Side chain Charge Transfer (PBS-CT) from the polypeptide backbone to the abundant cationic headgroups of Arginine in PRM, while the luminescence was believed to originate from charge recombination within the charge-separated excited states of PRM. Remarkably, since Arg is the only charged residue in the PRM sequence, the PRM Protein Charge Transfer Spectra (ProCharTS) is both totally and uniquely Arg specific. Interestingly, the peak of PRM luminescence emission spectrum was independent of the excitation wavelength, unlike other proteins such as human serum albumin, displaying unconventional luminescence. Aggregation-induced effects on PRM absorbance and luminescence were ruled out, as both PRM absorbance and luminescence increase maintained linearity with increasing concentration in the 25-150 µM range. Nucleoprotamine complex formation, resulting from the binding of PRM with calf-thymus genomic DNA (gDNA), was monitored through increased scattering by the nucleoprotamine complex, a decrease in gDNA/PRM absorbance, a decrease in gDNA/PRM ellipticity, and shifts of nucleoprotamine complex band upon agarose gel electrophoresis. Upon binding with gDNA (700 µM base pair concentration), PRM ProCharTS absorbance at 260 nm decreased by 72%. This decrease was attributed to the formation and subsequent precipitation of nucleoprotamine complex upon PRM-gDNA binding. The application of ProCharTS absorbance to indirectly monitor DNA-protein binding in a label-free approach was thus demonstrated.

Molecular and toxicological mechanisms behind the effects of chromium (VI) on the male reproductive system of Mytilus galloprovincialis: First evidence for poly-ADP-ribosylation of protamine-like II.

Studies on the molecular mechanisms of heavy metal toxicity in invertebrate reproduction are limited. Given that PARP-catalysed ADP-ribosylation is also involved in counteracting heavy metal toxicity and maintaining genomic integrity, and that PARylation is implicated in chromatin remodelling but its role in sperm chromatin remains to be elucidated, we investigated the effects of chromium(VI) at 1, 10 and 100 nM on the reproductive health of Mytilus galloprovincialis. The damage to the gonads was assessed by morphological analyses and the damage indices PARP and É£H2A.X were measured. Changes in the binding of protamine-like (PL) to DNA and the possibility of poly(ADP-ribosyl)ation of PL proteins were also investigated. Gonadal chromium accumulation and morphological damage were found, especially when the mussels were exposed to the highest dose of chromium(VI). In addition, the maximum expression of gonadal É£H2A.X and PARP were obtained at 100 and 10 nM Cr(VI), respectively. Interestingly, for the first time in all exposed conditions, poly(ADP)-ribosylation was detected on PL-II, which, together with PL-III and PL-IV, are the major nuclear basic proteins of Mytilus galloprovincialis sperm chromatin. Since PL-II is involved in the final high level of sperm chromatin compaction, this post-translational modification altered the binding of the PL protein to DNA, favouring the action of micrococcal nuclease on sperm chromatin. This study provides new insights into the effects of chromium(VI) on Mytilus galloprovincialis reproductive system and proposes a molecular mechanism hypothesis describing the toxic effects of this metal on PL-DNA binding, sperm chromatin and gonads.

Dual-mode upconversion sensors for detecting differently charged biotargets based on the oxidase-mimicking activity of Ce4+ and electrostatic control.

Heparin is a highly negatively charged sulfated linear polymer glycosaminoglycan that has been widely used as an anticoagulant in medicine. Protamine is a cationic protein rich in arginine that is used to treat the blood-brain barrier during excess heparin surgery. Trypsin is the most important digestive enzyme-encoding generated by the pancreas and can specifically cleave the carboxyl ends of arginine and lysine residues. Heparin, protamine, and trypsin interact and constrain each other, and their fluctuations reflect the body's dysfunction. Therefore, it is necessary to develop a fast, sensitive, and highly selective assay for regularly monitoring the levels of heparin, protamine, and trypsin in serum. Herein, a fluorescent and colorimetric dual-mode upconversion nanoparticle (UCNP) biosensor was used for the determination of heparin, protamine, and trypsin based on the oxidase-mimicking activity of Ce4+ and electrostatic control. The biosensor exhibited sensitive detection of heparin, protamine, and trypsin with low limits of detection (LODs) of 16 ng/mL, 87 ng/mL and 31 ng/mL, respectively. Furthermore, the designed biosensor could eliminate autofluorescence, which not only effectively increased the accuracy of the sensor but also provided a new sensing pathway for the detection of differently charged biotargets.

"Rich arginine and strong positive charge" antimicrobial protein protamine: From its action on cell membranes to inhibition of bacterial vital functions.

Protamine, an antimicrobial protein derived from salmon sperm with a molecular weight of approximately 5 kDa, is composed of 60-70 % arginine and is a highly charged protein. Here, we investigated the mechanism of antimicrobial action of protamine against Cutibacterium acnes (C. acnes) focusing on its rich arginine content and strong positive charge. Especially, we focused on the attribution of dual mechanisms of antimicrobial protein, including membrane disruption or interaction with intracellular components. We first determined the dose-dependent antibacterial activity of protamine against C. acnes. In order to explore the interaction between bacterial membrane and protamine, we analyzed cell morphology, zeta potential, membrane permeability, and the composition of membrane fatty acid. In addition, the localization of protamine in bacteria was observed using fluorescent-labeled protamine. For investigation of the intracellular targets of protamine, bacterial translation was examined using a cell-free translation system. Based on our results, the mechanism of the antimicrobial action of protamine against C. acnes is as follows: 1) electrostatic interactions with the bacterial cell membrane; 2) self-internalization into the bacterial cell by changing the composition of the bacterial membrane; and 3) inhibition of bacterial growth by blocking translation inside the bacteria. However, owing to its strong electric charge, protamine can also interact with DNA, RNA, and other proteins inside the bacteria, and may inhibit various bacterial life processes beyond the translation process.

Running the gauntlet: challenges to genome integrity in spermiogenesis.

Species' continuity depends on gametogenesis to produce the only cell types that can transmit genetic information across generations. Spermiogenesis, which encompasses post-meiotic, haploid stages of male gametogenesis, is a process that leads to the formation of sperm cells well-known for their motility. Spermiogenesis faces three major challenges. First, after two rounds of meiotic divisions, the genome lacks repair templates (no sister chromatids, no homologous chromosomes), making it incredibly vulnerable to any genomic insults over an extended time (typically days-weeks). Second, the sperm genome becomes transcriptionally silent, making it difficult to respond to new perturbations as spermiogenesis progresses. Third, the histone-to-protamine transition, which is essential to package the sperm genome, counterintuitively involves DNA break formation. How spermiogenesis handles these challenges remains poorly understood. In this review, we discuss each challenge and their intersection with the biology of protamines. Finally, we discuss the implication of protamines in the process of evolution.

Protamines and the sperm nuclear basic proteins Pandora's Box of insects.

Insects are the largest group of animals when it comes to the number and diversity of species. Yet, with the exception of Drosophila, no information is currently available on the primary structure of their sperm nuclear basic proteins (SNBPs). This paper represents the first attempt in this regard and provides information about six species of Neoptera: Poecillimon thessalicus, Graptosaltria nigrofuscata, Apis mellifera, Nasonia vitripennis, Parachauliodes continentalis, and Tribolium castaneum. The SNBPs of these species were characterized by acetic acid urea gel electrophoresis (AU-PAGE) and high-performance liquid chromatography fractionated. Protein sequencing was obtained using a combination of mass spectrometry sequencing, Edman N-terminal degradation sequencing and genome mining. While the SNBPs of several of these species exhibit a canonical arginine-rich protamine nature, a few of them exhibit a protamine-like composition. They appear to be the products of extensive cleavage processing from a precursor protein which are sometimes further processed by other post-translational modifications that are likely involved in the chromatin transitions observed during spermiogenesis in these organisms.

Ursonic acid attenuates spermatogenesis in oligozoospermia mice through inhibiting ferroptosis.

Ursonic acid (UNA) is a natural pentacyclic triterpene found in some medicinal plants and foods. The reproductive protective effect of UNA was evaluated in a mouse model of oligozoospermia induced by busulfan (BUS) at 30 mg/kg b.w.. The mice were initially divided into groups with UNA concentrations of 10, 30, 50, 100 mg/kg. Subsequently, based on sperm parameters, the optimal concentration of 50 mg/kg was identified. As a control, an additional group was supplemented with ursolic acid at a concentration of 50 mg/kg. The results indicated that BUS caused the loss of spermatogenic cells in testis, the decrease of sperm in epididymis, the disorder of testicular cytoskeleton, the decrease of serum sex hormones such as testosterone which induced an increase in feedback of androgen receptor and other testosterone-related proteins, the increase of malondialdehyde and reactive oxygen species levels and the increase of ferroptosis in testis while UNA successfully reversed these injuries. High-throughput sequencing revealed that UNA administration significantly upregulated the expression of genes associated with spermatogenesis, such as Tnp1, Tnp2, Prm1, among others. These proteins are crucial in the histone to protamine transition during sperm chromatin remodeling. Network pharmacology analysis reveals a close association between UNA and proteins related to the transformation of histones to protamine. Molecular docking studies reveal that UNA can interact with the ferroptosis-inhibiting gene SLC7A11, thereby modulating ferroptosis. Taken together, UNA alleviated BUS-induced oligozoospermia by regulating hormone secretion, mitigating oxidative stress and promoting recovery of spermatogenesis by inhibiting the ferroptosis.

Molecular and Functional Characterization of Human Sex-Determining Region on the Y Chromosome Variants Using Protamine 1 Promoter.

The male sex-determining gene, sex-determining region on the Y chromosome (SRY), is expressed in adult testicular germ cells; however, its role in regulating spermatogenesis remains unclear. The role of SRY in the postmeiotic gene expression was investigated by determining the effect of SRY on the promoter of the haploid-specific Protamine 1 (PRM1) gene, which harbors five distinct SRY-binding motifs. In a luciferase reporter assay system, SRY upregulates PRM1 promoter activity in vitro in a dose-dependent manner. Through a gel-shift assay involving a 31-bp DNA fragment encompassing the SRY element within the PRM1 promoter, the third SRY-binding site on the sense strand (-373/-367) was identified as crucial for PRM1 promoter activation. This assay was extended to analyze 9 SRY variants found in the testicular DNA of 44 azoospermia patients. The findings suggest that SRY regulates PRM1 promoter activity by directly binding to its specific motif within the PRM1 promoter.

Phase-separated CCER1 coordinates the histone-to-protamine transition and male fertility.

Idiopathic fertility disorders are associated with mutations in various genes. Here, we report that coiled-coil glutamate-rich protein 1 (CCER1), a germline-specific and intrinsically disordered protein (IDP), mediates postmeiotic spermatid differentiation. In contrast, CCER1 deficiency results in defective sperm chromatin compaction and infertility in mice. CCER1 increases transition protein (Tnp1/2) and protamine (Prm1/2) transcription and mediates multiple histone epigenetic modifications during the histone-to-protamine (HTP) transition. Immiscible with heterochromatin in the nucleus, CCER1 self-assembles into a polymer droplet and forms a liquid-liquid phase-separated condensate in the nucleus. Notably, we identified loss-of-function (LoF) variants of human CCER1 (hCCER1) in five patients with nonobstructive azoospermia (NOA) that were absent in 2713 fertile controls. The mutants led to premature termination or frameshift in CCER1 translation, and disrupted condensates in vitro. In conclusion, we propose that nuclear CCER1 is a phase-separated condensate that links histone epigenetic modifications, HTP transitions, chromatin condensation, and male fertility.

DNA groove preference shift upon phosphorylation of a protamine-like cationic peptide.

Protamines, arginine-rich DNA-binding proteins, are responsible for chromatin compaction in sperm cells, but their DNA groove preference, major or minor, is not clearly identified. We herein study the DNA groove preference of a short protamine-like cationic peptide before and after phosphorylation, using all-atom molecular dynamics and umbrella sampling simulations. According to various thermodynamic and structural analyses, a peptide in its non-phosphorylated native state prefers the minor groove over the major groove, but phosphorylation of the peptide bound to the minor groove not only reduces its binding affinity but also brings a serious deformation of the minor groove, eliminating the minor-groove preference. As protamines are heavily phosphorylated before binding to DNA, we expect that the structurally disordered phosphorylated protamines would prefer major grooves to enter into DNA during spermatogenesis.

Epigenetic aging of mammalian gametes.

The process of aging refers to physiological changes that occur to an organism as time progresses and involves changes to DNA, proteins, metabolism, cells, and organs. Like the rest of the cells in the body, gametes age, and it is well established that there is a decline in reproductive capabilities in females and males with aging. One of the major pathways known to be involved in aging is epigenetic changes. The epigenome is the multitude of chemical modifications performed on DNA and chromatin that affect the ability of chromatin to be transcribed. In this review, we explore the effects of aging on female and male gametes with a focus on the epigenetic changes that occur in gametes throughout aging. Quality decline in oocytes occurs at a relatively early age. Epigenetic changes constitute an important part of oocyte aging. DNA methylation is reduced with age, along with reduced expression of DNA methyltransferases (DNMTs). Histone deacetylases (HDAC) expression is also reduced, and a loss of heterochromatin marks occurs with age. As a consequence of heterochromatin loss, retrotransposon expression is elevated, and aged oocytes suffer from DNA damage. In sperm, aging affects sperm number, motility and fecundity, and epigenetic changes may constitute a part of this process. 5 methyl-cytosine (5mC) methylation is elevated in sperm from aged men, but methylation on Long interspersed nuclear elements (LINE) elements is reduced. Di and trimethylation of histone 3 lysine 9 (H3K9me2/3) is reduced in sperm from aged men and trimethylation of histone 3 lysine 27 (H3K27me3) is elevated. The protamine makeup of sperm from aged men is also changed, with reduced protamine expression and a misbalanced ratio between protamine proteins protamine P1 and protamine P2. The study of epigenetic reproductive aging is recently gaining interest. The current status of the field suggests that many aspects of gamete epigenetic aging are still open for investigation. The clinical applications of these investigations have far-reaching consequences for fertility and sociological human behavior.

Protamine folds DNA into flowers and loop stacks.

DNA in sperm undergoes an extreme compaction to almost crystalline packing levels. To produce this dense packing, DNA is dramatically reorganized in minutes by protamine proteins. Protamines are positively charged proteins that coat negatively charged DNA and fold it into a series of toroids. The exact mechanism for forming these ∼50-kbp toroids is unknown. Our goal is to study toroid formation by starting at the "bottom" with folding of short lengths of DNA that form loops and working "up" to more folded structures that occur on longer length scales. We previously measured folding of 200-300 bp of DNA into a loop. Here, we look at folding of intermediate DNA lengths (L = 639-3003 bp) that are 2-10 loops long. We observe two folded structures besides loops that we hypothesize are early intermediates in the toroid formation pathway. At low protamine concentrations (∼0.2 µM), we see that the DNA folds into flowers (structures with multiple loops that are positioned so they look like the petals of a flower). Folding at these concentrations condenses the DNA to 25% of its original length, takes seconds, and is made up of many small bending steps. At higher protamine concentrations (≥2 µM), we observe a second folded structure-the loop stack-where loops are stacked vertically one on top of another. These results lead us to propose a two-step process for folding at this length scale: 1) protamine binds to DNA, bending it into loops and flowers, and 2) flowers collapse into loop stacks. These results highlight how protamine uses a bind-and-bend mechanism to rapidly fold DNA, which may be why protamine can fold the entire sperm genome in minutes.

Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse protamine 1.

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. Phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues and their post-translational modifications are poorly understood. Here, we investigated the role of K49, a rodent-specific lysine residue in protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In sperm, alanine substitution (P1(K49A)) decreases sperm motility and male fertility-defects that are not rescued by arginine substitution (P1(K49R)). In zygotes, P1(K49A) leads to premature male pronuclear decompaction, altered DNA replication, and embryonic arrest. In vitro, P1(K49A) decreases protamine-DNA binding and alters DNA compaction and decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.

Ability of a selfish B chromosome to evade genome elimination in the jewel wasp, Nasonia vitripennis.

B chromosomes are non-essential, extra chromosomes that can exhibit transmission-enhancing behaviors, including meiotic drive, mitotic drive, and induction of genome elimination, in plants and animals. A fundamental but poorly understood question is what characteristics allow B chromosomes to exhibit these extraordinary behaviors. The jewel wasp, Nasonia vitripennis, harbors a heterochromatic, paternally transmitted B chromosome known as paternal sex ratio (PSR), which causes complete elimination of the sperm-contributed half of the genome during the first mitotic division of fertilized embryos. This genome elimination event may result from specific, previously observed alterations of the paternal chromatin. Due to the haplo-diploid reproduction of the wasp, genome elimination by PSR causes female-destined embryos to develop as haploid males that transmit PSR. PSR does not undergo self-elimination despite its presence with the paternal chromatin until the elimination event. Here we performed fluorescence microscopic analyses aimed at understanding this unexplained property. Our results show that PSR, like the rest of the genome, participates in the histone-to-protamine transition, arguing that PSR does not avoid this transition to escape self-elimination. In addition, PSR partially escapes the chromatin-altering activity of the intracellular bacterium, Wolbachia, demonstrating that this ability to evade chromatin alteration is not limited to PSR's own activity. Finally, we observed that the rDNA locus and other unidentified heterochromatic regions of the wasp's genome also seem to evade chromatin disruption by PSR, suggesting that PSR's genome-eliminating activity does not affect heterochromatin. Thus, PSR may target an aspect of euchromatin to cause genome elimination.

Arginine-rich peptides as crystallization inhibitors for sodium urate.

Inhibiting the formation of urate crystals is the key to prevent hyperuricemia from developing into gout. Although many studies have focused on the influence of biomacromolecules in the crystallization behavior of sodium urate, the role of peptides with specific structures may contribute to unprecedented regulatory effects. Here, for the first time, we studied the effects of cationic peptides on the phase behavior, crystallization kinetics, and size/morphology of urate crystals. The addition of protamine (PRTM, a typical natural arginine-rich peptide) prolongs the nucleation induction time of sodium urate and inhibits crystal nucleation effectively. PRTM binds to the surface of amorphous sodium urate (ASU) through the hydrogen bond and electrostatic attraction between guanidine groups and urate anions, which is conducive to maintaining the state of ASU and inhibiting crystal nucleation. Moreover, PRTM preferentially binds to the MSUM plane and leads to a significant reduction in the aspect ratio of MSUM filamentous crystals. Further studies showed that there are significant differences in the inhibiting effects of arginine-rich peptides with different chain lengths on the crystallization behavior of sodium urate. Both guanidine functional groups and peptide chain length determine the crystallization inhibiting effect of peptides simultaneously. The present work highlights the potential role of arginine peptides in inhibiting the crystallization of urate and provides new insights into the inhibition mechanism in the pathological biomineralization of sodium urate, demonstrating the possibility of using cationic peptides to treat gout.

Possible Molecular Mechanisms Underlying the Decrease in the Antibacterial Activity of Protamine-like Proteins after Exposure of Mytilus galloprovincialis to Chromium and Mercury.

Natural bioactive compounds represent a new frontier of antimicrobial molecules, and the marine ecosystem represents a new challenge in this regard. In the present work, we evaluated the possibility of changes in the antibacterial activity of protamine-like (PL) proteins, the major nuclear basic protein components of Mytilus galloprovincialis sperm chromatin, after the exposure of mussels to subtoxic doses of chromium (VI) (1, 10, and 100 nM) and mercury (1, 10, and 100 pM) HgCl2, since these metals affect some properties of PL. After exposure, we analyzed the electrophoretic pattern of PLs by both acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and determined the MIC and MBC of these proteins on different gram+ and gram- bacteria. PLs, particularly after mussels were exposed to the highest doses of chromium and mercury, showed significantly reduced antibacterial activity. Just at the highest doses of exposure to the two metals, changes were found in the electrophoretic pattern of PLs, suggesting that there were conformational changes in these proteins, which were confirmed by the fluorescence measurements of PLs. These results provide the first evidence of a reduction in the antibacterial activity of these proteins following the exposure of mussels to these metals. Based on the results, hypothetical molecular mechanisms that could explain the decrease in the antibacterial activity of PLs are discussed.

DOT1L promotes spermatid differentiation by regulating expression of genes required for histone-to-protamine replacement.

Unique chromatin remodeling factors orchestrate dramatic changes in nuclear morphology during differentiation of the mature sperm head. A crucial step in this process is histone-to-protamine exchange, which must be executed correctly to avoid sperm DNA damage, embryonic lethality and male sterility. Here, we define an essential role for the histone methyltransferase DOT1L in the histone-to-protamine transition. We show that DOT1L is abundantly expressed in mouse meiotic and postmeiotic germ cells, and that methylation of histone H3 lysine 79 (H3K79), the modification catalyzed by DOT1L, is enriched in developing spermatids in the initial stages of histone replacement. Elongating spermatids lacking DOT1L fail to fully replace histones and exhibit aberrant protamine recruitment, resulting in deformed sperm heads and male sterility. Loss of DOT1L results in transcriptional dysregulation coinciding with the onset of histone replacement and affecting genes required for histone-to-protamine exchange. DOT1L also deposits H3K79me2 and promotes accumulation of elongating RNA Polymerase II at the testis-specific bromodomain gene Brdt. Together, our results indicate that DOT1L is an important mediator of transcription during spermatid differentiation and an indispensable regulator of male fertility.

Derepression of Y-linked multicopy protamine-like genes interferes with sperm nuclear compaction in D. melanogaster.

Across species, sperm maturation involves the dramatic reconfiguration of chromatin into highly compact nuclei that enhance hydrodynamic ability and ensure paternal genomic integrity. This process is mediated by the replacement of histones by sperm nuclear basic proteins, also referred to as protamines. In humans, a carefully balanced dosage between two known protamine genes is required for optimal fertility. However, it remains unknown how their proper balance is regulated and how defects in balance may lead to compromised fertility. Here, we show that a nucleolar protein, modulo, a homolog of nucleolin, mediates the histone-to-protamine transition during Drosophila spermatogenesis. We find that modulo mutants display nuclear compaction defects during late spermatogenesis due to decreased expression of autosomal protamine genes (including Mst77F) and derepression of Y-linked multicopy Mst77F homologs (Mst77Y), leading to the mutant's known sterility. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear compaction defects, similar to modulo mutant, indicating that Mst77Y is a dominant-negative variant interfering with the process of histone-to-protamine transition. Interestingly, ectopic overexpression of Mst77Y caused decompaction of X-bearing spermatids nuclei more frequently than Y-bearing spermatid nuclei, although this did not greatly affect the sex ratio of offspring. We further show that modulo regulates these protamine genes at the step of transcript polyadenylation. We conclude that the regulation of protamines mediated by modulo, ensuring the expression of functional ones while repressing dominant-negative ones, is critical for male fertility.

Nucleosomes in mammalian sperm: conveying paternal epigenetic inheritance or subject to reprogramming between generations?

The genome of mammalian sperm is largely packaged by sperm-specific proteins termed protamines. The presence of some residual nucleosomes has, however, emerged as a potential source of paternal epigenetic inheritance between generations. Sperm nucleosomes bear important regulatory histone marks and locate at gene-regulatory regions, functional elements, and intergenic regions. It is unclear whether sperm nucleosomes are retained at specific genomic locations in a deterministic manner or are randomly preserved due to inefficient exchange of histones by protamines. Recent studies indicate heterogeneity in chromatin packaging within sperm populations and an extensive reprogramming of paternal histone marks post fertilization. Obtaining single-sperm nucleosome distributions is fundamental to estimating the potential of sperm-borne nucleosomes in instructing mammalian embryonic development and in the transmission of acquired phenotypes.