Evolutionary modes of wtf meiotic driver genes in Schizosaccharomyces pombe.

Killer meiotic drivers (KMDs) are a class of selfish genetic elements that bias inheritance in their favor by destroying meiotic progeny that do not carry them. How KMDs evolve is not well understood. In the fission yeast Schizosaccharomyces pombe, the largest gene family, known as the wtf genes, is a KMD family that causes intraspecific hybrid sterility. Here, we investigate how wtf genes evolve using long-read-based genome assemblies of 31 distinct S. pombe natural isolates, which encompass the known genetic diversity of S. pombe. Our analysis, involving nearly 1,000 wtf genes in these isolates, yields a comprehensive portrayal of the intraspecific diversity of wtf genes. Leveraging single-nucleotide polymorphisms in adjacent unique sequences, we pinpoint wtf-gene-containing loci that have recently undergone gene conversion events and infer their pre-gene-conversion state. These events include the revival of wtf pseudogenes, lending support to the notion that gene conversion plays a role in preserving this gene family from extinction. Moreover, our investigation reveals that solo long terminal repeats (LTRs) of retrotransposons, frequently found near wtf genes, can act as recombination arms, influencing the upstream regulatory sequences of wtf genes. Additionally, our exploration of the outer boundaries of wtf genes uncovers a previously unrecognized type of directly oriented repeats flanking wtf genes. These repeats may have facilitated the early expansion of the wtf gene family in S. pombe. Our findings enhance the understanding of the mechanisms influencing the evolution of this KMD gene family.

Fission yeast Duc1 links to ER-PM contact sites and influences PM lipid composition and cytokinetic ring anchoring.

Cytokinesis is the final stage of the cell cycle that results in the physical separation of daughter cells. To accomplish cytokinesis, many organisms build an actin- and myosin-based cytokinetic ring (CR) that is anchored to the plasma membrane (PM). Defects in CR-PM anchoring can arise when the PM lipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is depleted. In Schizosaccharomyces pombe, reduced PM PI(4,5)P2 results in a CR that cannot maintain a medial position and slides toward one cell end, resulting in two differently sized daughter cells. S. pombe PM PI(4,5)P2 is synthesized by the phosphatidylinositol 4-phosphate 5-kinase (PI5-kinase) Its3, but what regulates this enzyme to maintain appropriate PM PI(4,5)P2 levels in S. pombe is not known. To identify Its3 regulators, we used proximity-based biotinylation, and the uncharacterized protein Duc1 was specifically detected. We discovered that Duc1 decorates the PM except at the cell division site and that its unique localization pattern is dictated by binding to the endoplasmic reticulum (ER)-PM contact site proteins Scs2 and Scs22. Our evidence suggests that Duc1 also binds PI(4,5)P2 and helps enrich Its3 at the lateral PM, thereby promoting PM PI(4,5)P2 synthesis and robust CR-PM anchoring.

An improved tetracycline-inducible expression system for fission yeast.

The ability to manipulate gene expression is valuable for elucidating gene function. In the fission yeast Schizosaccharomyces pombe, the most widely used regulatable expression system is the nmt1 promoter and its two attenuated variants. However, these promoters have limitations, including a long lag, incompatibility with rich media, and unsuitability for non-dividing cells. Here, we present a tetracycline-inducible system free of these shortcomings. Our system features the enotetS promoter, which achieves a similar induced level and a higher induction ratio compared to the nmt1 promoter, without exhibiting a lag. Additionally, our system includes four weakened enotetS variants, offering an expression range similar to the nmt1 series promoters but with more intermediate levels. To enhance usability, each promoter is combined with a Tet-repressor-expressing cassette in an integration plasmid. Importantly, our system can be used in non-dividing cells, enabling the development of a synchronous meiosis induction method with high spore viability. Moreover, our system allows for the shutdown of gene expression and the generation of conditional loss-of-function mutants. This system provides a versatile and powerful tool for manipulating gene expression in fission yeast.

The Schizosaccharomyces pombe ornithine-N5-oxygenase Sib2 interacts with the N5-transacetylase Sib3 in the ferrichrome biosynthetic pathway.

The fission yeast Schizosaccharomyces pombe produces the hydroxamate-type siderophore ferrichrome (Fc). The biosynthesis of Fc requires the Fc synthase Sib1, the ornithine-N5-oxygenase Sib2, and the N5-hydroxyornithine-N5-transacetylase Sib3. In this study, we demonstrate the critical importance of the His248 residue of Sib3 in Fc production. Cells expressing a sib3H248A mutant allele fail to grow in iron-poor media without Fc supplementation. These sib3H248A mutant cells are consistently unable to promote Fc-dependent growth of Saccharomyces cerevisiae cells in cross-feeding experiments. Green fluorescent protein (GFP)-tagged wild-type Sib3 and mutant Sib3H248A exhibit a pancellular distribution. Coimmunoprecipitation assays revealed that both wild-type and Sib3H248A physically interact with Sib2. Further analysis identified a minimal C-terminal region from amino acids 290-334 of Sib3 that is required for interaction with Sib2. Deletion mapping analysis identified two regions of Sib2 as being required for its association with Sib3. The first region encompasses amino acids 1-135, and the second region corresponds to amino acids 281-358 of Sib2. Taken together, these results describe the first example of a physical interaction between an ornithine-N5-oxygenase and an N5-hydroxyornithine-N5-transacetylase controlling the biosynthesis of a hydroxamate-type siderophore.

Mild Heat Stress Alters the Physical State and Structure of Membranes in Triacylglycerol-Deficient Fission Yeast, Schizosaccharomyces pombe.

We investigated whether the elimination of two major enzymes responsible for triacylglycerol synthesis altered the structure and physical state of organelle membranes under mild heat shock conditions in the fission yeast, Schizosaccharomyces pombe. Our study revealed that key intracellular membrane structures, lipid droplets, vacuoles, the mitochondrial network, and the cortical endoplasmic reticulum were all affected in mutant fission yeast cells under mild heat shock but not under normal growth conditions. We also obtained direct evidence that triacylglycerol-deficient cells were less capable than wild-type cells of adjusting their membrane physical properties during thermal stress. The production of thermoprotective molecules, such as HSP16 and trehalose, was reduced in the mutant strain. These findings suggest that an intact system of triacylglycerol metabolism significantly contributes to membrane protection during heat stress.

Nitrogen signaling factor triggers a respiration-like gene expression program in fission yeast.

Microbes have evolved intricate communication systems that enable individual cells of a population to send and receive signals in response to changes in their immediate environment. In the fission yeast Schizosaccharomyces pombe, the oxylipin nitrogen signaling factor (NSF) is part of such communication system, which functions to regulate the usage of different nitrogen sources. Yet, the pathways and mechanisms by which NSF acts are poorly understood. Here, we show that NSF physically interacts with the mitochondrial sulfide:quinone oxidoreductase Hmt2 and that it prompts a change from a fermentation- to a respiration-like gene expression program without any change in the carbon source. Our results suggest that NSF activity is not restricted to nitrogen metabolism alone and that it could function as a rheostat to prepare a population of S. pombe cells for an imminent shortage of their preferred nutrients.

Analysis of transcriptional response in haploid and diploid Schizosaccharomyces pombe under genotoxic stress.

Whole genome duplications are implicated in genome instability and tumorigenesis. Human and yeast polyploids exhibit increased replication stress and chromosomal instability, both hallmarks of cancer. In this study, we investigate the transcriptional response of Schizosaccharomyces pombe to increased ploidy generally, and in response to treatment with the genotoxin methyl methanesulfonate (MMS). We find that treatment of MMS induces upregulation of genes involved in general response to genotoxins, in addition to cell cycle regulatory genes. Downregulated genes are enriched in transport and sexual reproductive pathways. We find that the diploid response to MMS is muted compared to the haploid response, although the enriched pathways remain largely the same. Overall, our data suggests that the global S. pombe transcriptome doubles in response to increased ploidy but undergoes modest transcriptional changes in both unperturbed and genotoxic stress conditions.

Understanding the molecular mechanisms of human diseases: the benefits of fission yeasts.

The role of model organisms such as yeasts in life science research is crucial. Although the baker's yeast (Saccharomyces cerevisiae) is the most popular model among yeasts, the contribution of the fission yeasts (Schizosaccharomyces) to life science is also indisputable. Since both types of yeasts share several thousands of common orthologous genes with humans, they provide a simple research platform to investigate many fundamental molecular mechanisms and functions, thereby contributing to the understanding of the background of human diseases. In this review, we would like to highlight the many advantages of fission yeasts over budding yeasts. The usefulness of fission yeasts in virus research is shown as an example, presenting the most important research results related to the Human Immunodeficiency Virus Type 1 (HIV-1) Vpr protein. Besides, the potential role of fission yeasts in the study of prion biology is also discussed. Furthermore, we are keen to promote the uprising model yeast Schizosaccharomyces japonicus, which is a dimorphic species in the fission yeast genus. We propose the hyphal growth of S. japonicus as an unusual opportunity as a model to study the invadopodia of human cancer cells since the two seemingly different cell types can be compared along fundamental features. Here we also collect the latest laboratory protocols and bioinformatics tools for the fission yeasts to highlight the many possibilities available to the research community. In addition, we present several limiting factors that everyone should be aware of when working with yeast models.

The fork protection complex promotes parental histone recycling and epigenetic memory.

The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.

Advanced Protocol for Molecular Characterization of Viral Genome in Fission Yeast (Schizosaccharomyces pombe).

Fission yeast, a single-cell eukaryotic organism, shares many fundamental cellular processes with higher eukaryotes, including gene transcription and regulation, cell cycle regulation, vesicular transport and membrane trafficking, and cell death resulting from the cellular stress response. As a result, fission yeast has proven to be a versatile model organism for studying human physiology and diseases such as cell cycle dysregulation and cancer, as well as autophagy and neurodegenerative diseases like Alzheimer's, Parkinson's, and Huntington's diseases. Given that viruses are obligate intracellular parasites that rely on host cellular machinery to replicate and produce, fission yeast could serve as a surrogate to identify viral proteins that affect host cellular processes. This approach could facilitate the study of virus-host interactions and help identify potential viral targets for antiviral therapy. Using fission yeast for functional characterization of viral genomes offers several advantages, including a well-characterized and haploid genome, robustness, cost-effectiveness, ease of maintenance, and rapid doubling time. Therefore, fission yeast emerges as a valuable surrogate system for rapid and comprehensive functional characterization of viral proteins, aiding in the identification of therapeutic antiviral targets or viral proteins that impact highly conserved host cellular functions with significant virologic implications. Importantly, this approach has a proven track record of success in studying various human and plant viruses. In this protocol, we present a streamlined and scalable molecular cloning strategy tailored for genome-wide and comprehensive functional characterization of viral proteins in fission yeast.

Fission yeast Pib2 localizes to vacuolar membranes and regulates TOR complex 1 through evolutionarily conserved domains.

TOR complex 1 (TORC1) is a multi-protein kinase complex that coordinates cellular growth with environmental cues. Recent studies have identified Pib2 as a critical activator of TORC1 in budding yeast. Here, we show that loss of Pib2 causes severe growth defects in fission yeast cells, particularly when basal TORC1 activity is diminished by hypomorphic mutations in tor2, the gene encoding the catalytic subunit of TORC1. Consistently, TORC1 activity is significantly compromised in the tor2 hypomorphic mutants lacking Pib2. Moreover, as in budding yeast, fission yeast Pib2 localizes to vacuolar membranes via its FYVE domain, with its tail motif indispensable for TORC1 activation. These results strongly suggest that Pib2-mediated positive regulation of TORC1 is evolutionarily conserved between the two yeast species.

The Mechanosensitive Pkd2 Channel Modulates the Recruitment of Myosin II and Actin to the Cytokinetic Contractile Ring.

Cytokinesis, the last step in cell division, separates daughter cells through mechanical force. This is often through the force produced by an actomyosin contractile ring. In fission yeast cells, the ring helps recruit a mechanosensitive ion channel, Pkd2, to the cleavage furrow, whose activation by membrane tension promotes calcium influx and daughter cell separation. However, it is unclear how the activities of Pkd2 may affect the actomyosin ring. Here, through both microscopic and genetic analyses of a hypomorphic pkd2 mutant, we examined the potential role of this essential gene in assembling the contractile ring. The pkd2-81KD mutation significantly increased the counts of the type II myosin heavy chain Myo2 (+18%), its regulatory light chain Rlc1 (+37%) and actin (+100%) molecules in the ring, compared to the wild type. Consistent with a regulatory role of Pkd2 in the ring assembly, we identified a strong negative genetic interaction between pkd2-81KD and the temperature-sensitive mutant myo2-E1. The pkd2-81KD myo2-E1 cells often failed to assemble a complete contractile ring. We conclude that Pkd2 modulates the recruitment of type II myosin and actin to the contractile ring, suggesting a novel calcium-dependent mechanism regulating the actin cytoskeletal structures during cytokinesis.

Arsenite treatment induces Hsp90 aggregatesdistinct from conventional stress granules in fission yeast.

Various stress conditions, such as heat stress (HS) and oxidative stress, can cause biomolecular condensates represented by stress granules (SGs) via liquid-liquid phase separation. We have previously shown that Hsp90 forms aggregates in response to HS and that Hsp90 aggregates transiently co-localize with SGs as visualized by Pabp. Here, we showed that arsenite, one of the well-described SG-inducing stimuli, induces Hsp90 aggregates distinct from conventional SGs in fission yeast. Arsenite induced Hsp90 granules in a dose-dependent manner, and these granules were significantly diminished by the co-treatment with a ROS scavenger N-acetyl cysteine (NAC), indicating that ROS are required for the formation of Hsp90 granules upon arsenite stress. Notably, Hsp90 granules induced by arsenite do not overlap with conventional SGs as represented by eIF4G or Pabp, while HS-induced Hsp90 granules co-localize with SGs. Nrd1, an RNA-binding protein known as a HS-induced SG component, was recruited into Hsp90 aggregates but not to the conventional SGs upon arsenite stress. The non-phosphorylatable eIF2α mutants significantly delayed the Hsp90 granule formation upon arsenite treatment. Importantly, inhibition of Hsp90 by geldanamycin impaired the Hsp90 granule formation and reduced the arsenite tolerance. Collectively, arsenite stimulates two types of distinct aggregates, namely conventional SGs and a novel type of aggregates containing Hsp90 and Nrd1, wherein Hsp90 plays a role as a center for aggregation, and stress-specific compartmentalization of biomolecular condensates.

Cytoplasmic fluidization contributes to breaking spore dormancy in fission yeast.

The cytoplasm is a complex, crowded environment that influences myriad cellular processes including protein folding and metabolic reactions. Recent studies have suggested that changes in the biophysical properties of the cytoplasm play a key role in cellular homeostasis and adaptation. However, it still remains unclear how cells control their cytoplasmic properties in response to environmental cues. Here, we used fission yeast spores as a model system of dormant cells to elucidate the mechanisms underlying regulation of the cytoplasmic properties. By tracking fluorescent tracer particles, we found that particle mobility decreased in spores compared to vegetative cells and rapidly increased at the onset of dormancy breaking upon glucose addition. This cytoplasmic fluidization depended on glucose-sensing via the cyclic adenosine monophosphate-protein kinase A pathway. PKA activation led to trehalose degradation through trehalase Ntp1, thereby increasing particle mobility as the amount of trehalose decreased. In contrast, the rapid cytoplasmic fluidization did not require de novo protein synthesis, cytoskeletal dynamics, or cell volume increase. Furthermore, the measurement of diffusion coefficients with tracer particles of different sizes suggests that the spore cytoplasm impedes the movement of larger protein complexes (40 to 150 nm) such as ribosomes, while allowing free diffusion of smaller molecules (~3 nm) such as second messengers and signaling proteins. Our experiments have thus uncovered a series of signaling events that enable cells to quickly fluidize the cytoplasm at the onset of dormancy breaking.

Ssu72 phosphatase deficiency leads to spindle crossing during the second meiotic division process.

Ssu72 is a component of the yeast cleavage/polyadenylation factor (CPF) complex, which catalyzes the dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II at S5-P and S7-P. It has been shown that Ssu72 phosphatase is involved in regulating chromosome cohesion during mitosis. To further clarify whether Ssu72 phosphatase affects chromosome separation during meiotic division in Schizosaccharomyces pombe, we utilized green fluorescent protein (GFP) to label centromeres and red fluorescent protein to label microtubule protein Atb2. The entire meiotic chromosome separation process of ssu72∆ cells was observed in real-time under fluorescence microscope. It was found that two spindles of ssu72∆ cells crossed during the metaphase and anaphase of the second meiotic division, and this spindle crossing led to a new type of spore defect distribution pattern. The results of this study can provide important reference significance for studying the roles of phosphatase Ssu72 in higher organisms.

Non-Mitochondrial Aconitase-2 Mediates the Transcription of Nuclear-Encoded Electron Transport Chain Genes in Fission Yeast.

Aconitase-2 (Aco2) is present in the mitochondria, cytosol, and nucleus of fission yeast. To explore its function beyond the well-known role in the mitochondrial tricarboxylic acid (TCA) cycle, we conducted genome-wide profiling using the aco2ΔNLS mutant, which lacks a nuclear localization signal (NLS). The RNA sequencing (RNA-seq) data showed a general downregulation of electron transport chain (ETC) genes in the aco2ΔNLS mutant, except for those in the complex II, leading to a growth defect in respiratory-prone media. Complementation analysis with non-catalytic Aco2 [aco2ΔNLS + aco2(3CS)], where three cysteines were substituted with serine, restored normal growth and typical ETC gene expression. This suggests that Aco2's catalytic activity is not essential for its role in ETC gene regulation. Our mRNA decay assay indicated that the decrease in ETC gene expression was due to transcriptional regulation rather than changes in mRNA stability. Additionally, we investigated the Php complex's role in ETC gene regulation and found that ETC genes, except those within complex II, were downregulated in php3Δ and php5Δ strains, similar to the aco2ΔNLS mutant. These findings highlight a novel role for nuclear aconitase in ETC gene regulation and suggest a potential connection between the Php complex and Aco2.

Suppression of inositol pyrophosphate toxicosis and hyper-repression of the fission yeast PHO regulon by loss-of-function mutations in chromatin remodelers Snf22 and Sol1.

Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes pho1, pho84, and tgp1) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP8 levels derepress the PHO regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP8 are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP8 toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP8 toxicity is alleviated by deletion of snf22+, the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating snf22-(D996A-E997A) allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of snf22+ hyper-repressed pho1 expression in phosphate-replete cells; suppressed the pho1 derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious prt lncRNA termination); and delayed pho1 induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the tgp1 and pho1 promoters, but not for the promoters that drive the synthesis of the PHO-repressive lncRNAs. Transcription profiling of snf22∆ and snf22-(D996A-E997A) cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic asp1-STF alleles derepress the PHO genes via the action of IP8 as an agonist of precocious lncRNA 3'-processing/termination. IP8 toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP8 levels on termination. In this study, a forward genetic screen revealed that IP8 toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the PHO mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of PHO-activating transcription factor Pho7 to its binding sites in the PHO mRNA promoters.

Disordered region of nuclear membrane protein Bqt4 recruits phosphatidic acid to the nuclear envelope to maintain its structural integrity.

The nuclear envelope (NE) is a permeable barrier that maintains nuclear-cytoplasmic compartmentalization and ensures nuclear function; however, it ruptures in various situations such as mechanical stress and mitosis. Although the protein components for sealing a ruptured NE have been identified, the mechanism by which lipid components are involved in this process remains to be elucidated. Here, we found that an inner nuclear membrane (INM) protein Bqt4 directly interacts with phosphatidic acid (PA) and serves as a platform for NE maintenance in the fission yeast Schizosaccharomyces pombe. The intrinsically disordered region (IDR) of Bqt4, proximal to the transmembrane domain, binds to PA and forms a solid aggregate in vitro. Excessive accumulation of Bqt4 IDR in INM results in membrane overproliferation and lipid droplet formation in the nucleus, leading to centromere dissociation from the NE and chromosome missegregation. Our findings suggest that Bqt4 IDR controls nuclear membrane homeostasis by recruiting PA to the INM, thereby maintaining the structural integrity of the NE.

Rcn1, the fission yeast homolog of human DSCR1, regulates arsenite tolerance independently from calcineurin.

Calcineurin (CN) is a conserved Ca2+/calmodulin-dependent phosphoprotein phosphatase that plays a key role in Ca2+ signaling. Regulator of calcineurin 1 (RCAN1), also known as Down syndrome critical region gene 1 (DSCR1), interacts with calcineurin and inhibits calcineurin-dependent signaling in various organisms. Ppb1, the fission yeast calcineurin regulates Cl--homeostasis, and Ppb1 deletion induces MgCl2 hypersensitivity. Here, we characterize the conserved and novel roles of the fission yeast RCAN1 homolog rcn1+. Consistent with its role as an endogenous calcineurin inhibitor, Rcn1 overproduction reproduced the calcineurin-null phenotypes, including MgCl2 hypersensitivity and inhibition of calcineurin signaling upon extracellular Ca2+ stimuli as evaluated by the nuclear translocation and transcriptional activation of the calcineurin substrate Prz1. Notably, overexpression of rcn1+ causes hypersensitivity to arsenite, whereas calcineurin deletion induces arsenite tolerance, showing a phenotypic discrepancy between Rcn1 overexpression and calcineurin deletion. Importantly, although Rcn1 deletion induces modest sensitivities to arsenite and MgCl2 in wild-type cells, the arsenite tolerance, but not MgCl2 sensitivity, associated with Ppb1 deletion was markedly suppressed by Rcn1 deletion. Collectively, our findings reveal a previously unrecognized functional collaboration between Rcn1 and calcineurin, wherein Rcn1 not only negatively regulates calcineurin in the Cl- homeostasis, but also Rcn1 mediates calcineurin signaling to modulate arsenite cytotoxicity.

Characterization and quantitation of a sulfoconjugated metabolite for detection of methyltestosterone misuse and direct identification by LC-MS.

Methyltestosterone (MT) is one of the most frequently misused anabolic androgenic steroids detected in doping control analysis. The metabolism of MT in humans leads to several phase І metabolites and their corresponding phase Ⅱ conjugates. Previous studies have postulated the 3α-sulfoconjugate of 17α-methyl-5ß-androstane-3α,17ß-diol (S2) as principal sulfate metabolite of MT, with a detection window exceeding 10 days. However, a final direct and unambiguous confirmation of the structure of this metabolite is missing until now. In this study, we established an approach to detect and identify S2, using intact analysis by liquid chromatography hyphenated with tandem mass spectrometry (LC-MS/MS) without complex sample pretreatment. An in vitro study yielded the LC-MS/MS reference retention times of all 3-sulfated 17-methylandrostane-3,17-diol diastereomers, allowing for accurate structure assignment of potentially detected metabolites. In an in vivo excretion study with a single healthy male volunteer, the presence of the metabolite S2 was confirmed after a single oral dose of 10 mg MT. The reference standard was chemically synthesized, characterized by accurate mass mass spectrometry (MS) and nuclear magnetic resonance (NMR), and quantified by quantitative NMR (qNMR). Thus, this study finally provides accurate structure information on the S2 metabolite and a direct analytical method for detection of MT misuse. The availability of the reference material is expected to facilitate further evaluation and subsequent analytical method validation in anti-doping research.