RESEARCH ARTICLE Human sperm displays rapid responses to diet Daniel Nätt ID1☯*, Unn Kugelberg1☯, Eduard Casas ID2, Elizabeth Nedstrand3, Stefan Zalavary ID3, Pontus Henriksson ID4,5, Carola Nijm3, Julia Jäderquist3, Johanna Sandborg ID4,5, Eva Flinke5, Rashmi Ramesh1, Lovisa Örkenby1, Filip Appelkvist1, Thomas Lingg1, Nicola Guzzi6, Cristian Bellodi ID6, Marie Löf ID4,5, Tanya Vavouri ID2, Anita Öst ID1* 1 Linköping University, Department of Clinical and Experimental Medicine, Division of Neurobiology, a1111111111 Linkoping, Sweden, 2 Josep Carreras Leukaemia Research Institute (IJC), Program for Predictive and a1111111111 Personalized Medicine of Cancer (PMPPC-IGTP), Barcelona, Spain, 3 Linköping University, Department of Clinical and Experimental Medicine, Division of Obstetrics and Gynecology, Linköping, Sweden, 4 Karolinska a1111111111 Institute, Department of Biosciences and Nutrition, Huddinge, Sweden, 5 Linköping University, Department of a1111111111 Medical and Health Sciences, Division of Community Medicine, Linköping, Sweden, 6 Lund University, Stem a1111111111 Cell Center, Department of Laboratory Medicine, Division of Molecular Hematology, Lund, Sweden ☯ These authors contributed equally to this work. * daniel.natt@liu.se (DN); anita.ost@liu.se (AO) OPEN ACCESS Citation: Nätt D, Kugelberg U, Casas E, Nedstrand Abstract E, Zalavary S, Henriksson P, et al. (2019) Human sperm displays rapid responses to diet. PLoS Biol The global rise in obesity and steady decline in sperm quality are two alarming trends that 17(12): e3000559. https://doi.org/10.1371/journal. have emerged during recent decades. In parallel, evidence from model organisms shows pbio.3000559 that paternal diet can affect offspring metabolic health in a process involving sperm tRNA- Academic Editor: Jason W. Locasale, Duke derived small RNA (tsRNA). Here, we report that human sperm are acutely sensitive to nutri- University, UNITED STATES ent flux, both in terms of sperm motility and changes in sperm tsRNA. Over the course of a Received: May 16, 2019 2-week diet intervention, in which we first introduced a healthy diet followed by a diet rich in Accepted: November 18, 2019 sugar, sperm motility increased and stabilized at high levels. Small RNA-seq on repeatedly Published: December 26, 2019 sampled sperm from the same individuals revealed that tsRNAs were up-regulated by eat- ing a high-sugar diet for just 1 week. Unsupervised clustering identified two independent Copyright: © 2019 Nätt et al. This is an open access article distributed under the terms of the pathways for the biogenesis of these tsRNAs: one involving a novel class of fragments with Creative Commons Attribution License, which specific cleavage in the T-loop of mature nuclear tRNAs and the other exclusively involving permits unrestricted use, distribution, and mitochondrial tsRNAs. Mitochondrial involvement was further supported by a similar up-reg- reproduction in any medium, provided the original ulation of mitochondrial rRNA-derived small RNA (rsRNA). Notably, the changes in sugar- author and source are credited. sensitive tsRNA were positively associated with simultaneous changes in sperm motility Data Availability Statement: All relevant data and negatively associated with obesity in an independent clinical cohort. This rapid generated during the current study are contained within the paper and its Supporting Information response to a dietary intervention on tsRNA in human sperm is attuned with the paternal files, with one exception. S1 Fig was generated intergenerational metabolic responses found in model organisms. More importantly, our from fastq sequence files, which contain personal findings suggest shared diet-sensitive mechanisms between sperm motility and the biogen- genetic information. Unrestricted distribution of esis of tsRNA, which provide novel insights about the interplay between nutrition and male these files is therefore risking the personal integrity of the study participants, which violates our current reproductive health. ethical permits, participants consents, and Swedish laws. Nevertheless, these files may become available on request, given that the researcher adhere to the existing ethical permits and consents, or extend the ethical permits and consents by PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 1 / 25 Human sperm displays rapid responses to diet applying for this at the Swedish Ethical Review Introduction Authority (more information on how to apply contact, registrator@etikprovning.se, or visit Epidemiological studies have for decades reported worldwide declines in sperm quality among https://etikprovningsmyndigheten.se/). For specific healthy men [1–3]. While interpretations of these studies, which sometimes reach apocalyptic information about the data, please contact the proportions, are rightly criticized for often being underpowered, regionally dependent, and corresponding authors at daniel.natt@liu.se, anita. biased by covariates (for example see [4]), the consistency is reason enough for concern. A natt@liu.se. Data used in the current study, but recent meta-analysis of 137 reports estimated a 57% decline in sperm concentration during the generated by others, have previously been deposited at the Sequence Read Archive (SRA) and past 35 years, where best support for such declines was found in North America, Europe, and can be accessed through these accession Asia [5]. Ongoing large-scale studies, covering tens to hundreds of thousands of individuals, numbers: SRP065418, SRP132262 (a link is also also suggest that this decline shows no sign of recovery [6–8]. Thus, it has become increasingly provided to Gene Expression Omnibus by these urgent to better understand factors that affect sperm quality in humans. accessions GSE74426, GSE110190). Risk factors for low sperm quality in healthy men involve, for example, male reproductive Funding: The study was kindly supported by age, environmental exposures to endocrine disruptors (e.g., pesticides and heavy metals), and grants from The Swedish Research Council (2015- lifestyle factors (e.g., tobacco/alcohol and exercise) [5]. Obesity—with associated pathologies 03141; https://www.vr.se/english.html; received by such as diabetes—is also a strong risk factor [9–12]. Interestingly, many of the most frequently AÖ), Knut and Alice Wallenberg Foundation (Wallenberg Academy Fellow, 2015.0165; https:// studied populations with declines in sperm quality have also experienced recent rises in obe- kaw.wallenberg.org/wallenberg-academy-fellows; sity. It is well known that nutritional and metabolic factors may affect male fertility [9,13,14], received by AÖ), Ragnar Söderberg (Fellow in but little is known about the molecular mechanisms. Clues may, however, be found in recent Medicine 2015; https://ragnarsoderbergsstiftelse. discoveries of so-called paternal intergenerational metabolic responses in animals. se/; received by AÖ), Strategic Research Area In paternal intergenerational metabolic responses, males are exposed to dietary interven- Health Care Science at Karolinska Institutet/Umeå tions that create robust metabolic ripples that propagate through one or two generations University (https://ki.se/en/research/strategic- research-area-health-care-science-sfo-v; received before subsiding [15,16]. Such phenomena have been observed in many organisms, including by ML). The funders had no role in study design, humans, mice, and fruit flies [17–19]. The best candidate mechanism here involves changes in data collection and analysis, decision to publish, or the sperm load of small noncoding RNA (sncRNA). In general, RNAs are known to play essen- preparation of the manuscript. tial roles in establishing epigenetic states, including centromeric heterochromatin [20] and Competing interests: The authors have declared transposon silencing in the germ cells [21]. A subtype of sncRNA, tRNA-derived small RNAs that no competing interests exist. (tsRNAs), are known to be abundant in mammalian sperm—including human—and are play- Abbreviations: BMI, body mass index; BMR, basal ing a role in paternal intergenerational metabolic responses in mice [22–28]. The functional metabolic rate; FDR, False Discovery Rate; GEO, significance of tRNA fragments is just being unraveled but has so far been implicated in inhibi- Gene Expression Omnibus; hESC, human tion of translation, stress granule formation, and control of retrotransposons [24,29–31]. embryonic stem cell; lincRNA, long non-coding Whether tsRNA of human sperm is responsive to dietary interventions, and whether it associ- RNA; miRNA, microRNA; nitRNA, nuclear internal ates with changes in sperm quality, have not been investigated. T-loop tsRNA; PAL, physical activity level; piRNA, piwi interacting RNA; PUS7, Pseudouridine Here, we present the acute effects on human sperm following a 2-step diet intervention. Synthase 7; qPCR, quantitative PCR; RDI, This intervention involved, first, 1 week of healthy diet, to establish a baseline, followed by 1 recommended daily intake; RPM, reads per million; week of additional sugar intake on top of that. By investigating 3 ejaculates from the same indi- rsRNA, rRNA-derived small RNA; RT, retro- viduals, we found that sperm motility dramatically stabilized at high levels in all individuals transcription; sncRNA, small noncoding RNA; SRA, during the intervention. Changes in sperm motility were paralleled with a simultaneous Sequence Read Archive; TEE, total energy increase of tsRNA, primarily from mitochondrial origin, but also of a specific type of nuclear expenditure; tsRNA, tRNA-derived small RNA. tsRNA. These nuclear tsRNAs, which we name nuclear internal T-loop tsRNA (nitRNA), had a specific cut-site within the conserved TψC region in the T-loop of mature tRNA, indicative of a sugar-sensitive enzyme promoting the biogenesis of this tsRNA subtype. Thus, the sncRNA repertoire in human sperm, as well as sperm motility, show a fast and highly specific response to dietary changes. Results Changes in diet provoke rapid systemic responses To examine the responsiveness of human sperm to dietary changes, we recruited 15 healthy men—nonsmokers, 20–27 years old with a normal body mass index (BMI)—and subjected PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 2 / 25 Human sperm displays rapid responses to diet Fig 1. A 2-step diet intervention results in systemic metabolic responses and increased sperm motility. (A) Participants (n = 15) were given a highly controlled standard diet for 1 week (100% of RDI based on their TEE), followed by a week with additional sugar (+50% of RDI). (B) Changes in fat and fat free mass were estimated by BodPod measurements. (C) Serum triglycerides during the test period. (D) Semen was collected from each participant at the beginning of the study and at the end of each week. (E) Sperm motility during the test period. Data are available in S2 Table. RDI, recommended daily intake; TEE, total energy expenditure. https://doi.org/10.1371/journal.pbio.3000559.g001 them to a personalized dietary regime (Fig 1A). All participants agreed to only consume meals provided by the research team for the 2-week intervention. During the first week, we provided each participant with a healthy diet, according to the Nordic nutrient recommendation [32], with a total energy content corresponding to their estimated total energy expenditure (TEE) (S1 Table). The second week, their diet was supplemented with sugar corresponding to an additional 50% of their estimated TEE (average of 375 g sugar/day, equivalent to approxi- mately 3.5 L of sugar-sweetened beverages or approximately 450 g of candy). This 2-step strat- egy generated paired sample timelines that enabled each individual to be their own control. The caloric content of the first week’s diet was calculated to maintain the initial weight, whereas the caloric intake of the second week was estimated to increase the individual’s weight by 1.5 kg. As an indicator of adherence to the diets, the weight changes met these expectations (S2 Table). The ratio of fat versus lean mass showed that a major proportion of the increased weight was lean mass (Fig 1B), indicating that a sugar-rich diet in healthy, young men has an anabolic effect in the short term. Blood samples were collected at each time point, and whereas there were small but significant shifts in several parameters—including hemoglobin, thrombo- cyte concentration and glutamyl transferase (S2 Table)—there were prominent changes in serum triglyceride (Fig 1C) as well as a clear shift in cholesterol metabolism (S2 Table). It is worth noticing that fasting blood glucose was unaffected by 1 week of high-sugar diet, con- firming nonpathological sugar metabolism in the participants (S2 Table). Semen samples were collected 3 times: first at the start of the intervention, second after the healthy diet week, and third after the week with a high-sugar diet (Fig 1D). Total number of sperms was variable between participants and was unaffected by diet (S2 Table). We noticed, PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 3 / 25 Human sperm displays rapid responses to diet however, that sperm motility was steadily stabilized at high levels in all individuals during the 2 weeks of intervention (black line Fig 1E). Looking at the individual timelines, it was notice- able that 5 of 15 participants had a very low starting point, close to or under the reference value of 34% (S2 Table). By plotting this low sperm-motility group separately, it was clear that this group was remarkably improving their sperm motility during the test period, with the most pronounced effect already apparent after the first week (gray lines Fig 1E). In summary, the simultaneous increase of sperm motility and lean mass indicates that 1 week of a high-sugar diet on top of a healthy baseline had an anabolic effect in our young, healthy, and lean participants. Dietary sugar acutely modulates tsRNA in sperm Next, we extracted RNA from the sperm samples and performed small RNA sequencing. Our analytical workflow allowed for 16–45 nucleotides small RNA to be analyzed. Size distribution and first nucleotide bias were strongly conserved across all 3 ejaculates from the same individ- ual (S1 Fig), which validates the integrity of our experiment. In concordance with earlier reports [22,25], the majority of small RNAs in human sperm were identified as tsRNA, rRNA- derived small RNA (rsRNA), and microRNA (miRNA) (Fig 2A/2B) (S1 Data). The 2-step diet intervention was primarily designed to examine a high-sugar diet in relation to a healthy, baseline diet (Fig 2C). We therefore first focused on this comparison. One week of high-sugar diet induced a marked shift in the relative distribution of small RNAs, such that miRNA and tsRNA (primarily of mitochondrial origin) increased while ribosomal-derived small RNA (rsRNA) decreased (Fig 2D). Individual miRNAs, however, did not change signifi- cantly following a high-sugar diet (S3 Table; False Discovery Rate [FDR] corrected). Closer examinations of the rsRNA revealed that the down-regulation was due to massive small-effect changes in the highly expressed 18S and 28S subunits (S2 Fig). These rsRNAs compromised 92.6% of all rsRNA in our data and have nuclear origins. More intriguingly, the far less expressed 12S and 16S derived rsRNA transcribed from the mitochondrial genome were sig- nificantly up-regulated (S2 Fig). Focusing on tsRNA, we next annotated the complex mix of tsRNAs into 5 subtypes: 50 -half, 5 -tsRNA, i-tsRNA, 30 -tsRNA and 30 -half (Fig 2E) [33]. Using this approach, it was clear that 0 50 -fragments were most abundant, as previously shown in mice [26] and that 30 -fragments were least abundant, whereas the less studied i-tsRNAs were present at intermediate levels (Fig 2F). Only i-tsRNAs and 30 -tsRNAs, however, exhibited significant changes in response to the high-sugar diet (Fig 2G). Focusing on tRNA isodecoders, we found that tsRNA from 8 isode- coders changed upon a high-sugar diet intervention (Fig 2H, middle panel). These 8 isodeco- ders were moderately expressed (Fig 2H, left panel) and had a high content of i-tsRNAs or 30 - tsRNAs (Fig 2H, right panel). In contrast, highly abundant tRNA isodecoders, such as GlyGCC, GlyCCC, and GluCTC (Fig 2H, left panel), were unaffected by the high-sugar diet (Fig 2H, middle panel) and had a high content of 50 -tsRNAs (Fig 2H, right panel). To validate these results, we did quantitative PCR (qPCR) on 2 identified tsRNA, the nuclear LysCTT i-tsRNA and mitochondrial SerTGA 50 tsRNA (S3 Fig). Both sperm and human embryonic stem cells (hESCs) generated unique fragments in the expected sizes after qPCR. Most importantly, sequencing and qPCR results correlated well across all participants, and the effect of sugar was validated. In summary, specific subtypes of sperm tsRNAs, primarily i-tsRNAs and 30 -tsRNAs, are acutely up-regulated in response to diet. Notably, this involved tsRNA from both nuclear as well as mitochondrial tRNA. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 4 / 25 Human sperm displays rapid responses to diet Fig 2. Human sperm tRNA fragments are acutely sensitive to high-sugar diet. (A) Small RNA-seq profiles from motile sperm of each participant (S1-S16; n = 15) were analyzed at the experimental start point (“Start”), after the first week of healthy diet (“Healthy”), and after the second week of high-sugar diet (“Sugar”). (B) Mean proportion of small RNA across the experiment. (C) The intervention was primarily designed for investigating the effect of Sugar compared to Healthy (baseline). (D) Fold changes in biotypes after 1 week of high-sugar diet (Sugar/Healthy). (E) Types of tsRNA analyzed. (F) Mean number of reads for the different types of tsRNAs. (G) Fold changes of tsRNA types PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 5 / 25 Human sperm displays rapid responses to diet after 1 week of high-sugar diet. (H) Top expressed nuclear and mitochondrial tRNA isodecoders, their mean expression, fold change after 1 week of high-sugar diet, and their composition of tsRNA types. Only tRNA isodecoders with at least 100 RPM are presented. Error bars indicate ± SEM. “� ” Indicates at least p < 0.05. Graphs can be reproduced using the script in S1 Text with input from S1 and S2 Data. FC, fold change; lincRNA, long non-coding RNA; miRNA, microRNA; Mt, Mitochondrial; piRNA, piwi interacting RNA; RPM, reads per million; rsRNA, rRNA-derived small RNA; tsRNA, tRNA-derived small RNA. https://doi.org/10.1371/journal.pbio.3000559.g002 Mitochondrial and nuclear tsRNA form distinct clusters For each sugar-sensitive tRNA isodecoder (Fig 2H), several tsRNAs were identified, many dif- fering in just one or a few nucleotides (S4 Table, S2 Data), most likely indicating common pro- cessing pathways. To examine this, we used unsupervised clustering to reveal the underlying correlational structure of these tsRNAs. As suspected, the expressional profiles of tRNA isode- coder subtype were strongly related across individuals (Fig 3). More importantly, nuclear and mitochondrial tsRNA formed 2 completely separate clusters. While the nuclear cluster exclusively contained i-tsRNA, the mitochondrial cluster primarily contained a mixture of i- tsRNA and 30 -tsRNA. This indicates that the discrepancy between i-tsRNA and 30 -tsRNA, reported above, are generated by at least 2 processing pathways separated by cellular compartmentation. Mitochondrial mixed i N uc 5' HisGTG HisGTG HisGT le i G ThrT A C HisGT ar 3' Ser GA Th TGT A Se Va hrT T ValT 3' T rTG TG GT Se rT G lT GT Thr 5 6 7 8 Le rT 9 AC 4 Le 3 10 GA uC AC 11 A r uC A 2 12 lT C A 1 gC A 54 13 a A Ly C 14 V lTA T sC G 53 15 Va Arg T T 52 rTG Th i GTG 16 CCT 51 Le uC 17 His AA 50 TG 18 HisG ArgCCG 49 HisGTG 19 48 20 LeuCAA 47 ThrTGT 3' 21 ArgCCG 46 ArgCCT 22 AA 45 ArgC Le uC 23 CT L 24 ysC CCG 44 TT Arg CG 43 25 A rgC C 26 Ly CT Arg CCG 41 42 L 27 y s CT g G 40 sC T Ar CC 28 TT Ly s C T gC G g 39 29 Ar Ar CC 38 Ly s C T gC G s C TT 30 37 Ly C TT Arg CT C 36 35 31 g 34 33 32 TT Ly s TT ArgC CG Ar Ly s C CT Ly s C T LysCTT T LysCTT Ly s C T C Ar T i Nuclear i-tsRNA Fig 3. Mitochondrial and nuclear tsRNA form distinct clusters. Dendrogram shows the relatedness of the regulatory responses of individual tsRNAs from the significantly changed tRNA isodecoders in (Fig 2H). Each leaf represents the differences between Sugar and Healthy in a single tsRNA across the 15 participants. Mitochondrial (red) and nuclear i- tsRNAs (blue) shows clear division into 2 clusters. Hierarchical cluster analysis was based on Euclidean distances across center scaled RPM differences between the Sugar and the Healthy sample of each participant. For more information about these tsRNAs, see S5 Table. The graph can be reproduced using the script in S1 Text with input from S2 Data. RPM, reads per million; tsRNA, tRNA-derived small RNA. https://doi.org/10.1371/journal.pbio.3000559.g003 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 6 / 25 Human sperm displays rapid responses to diet Obese men have altered sperm tsRNA To test the integrity of our findings, we next reanalyzed data from a Danish study by Donkin and colleagues [25], which sequenced sperm small RNA from lean and clinically obese individ- uals (Fig 4A). Intriguingly, by replicating our analytical workflow on this dataset, we found i- tsRNAs to be the only tsRNA subtype that differed significantly between these two groups (Fig 4B). Comparing the sugar-sensitive tsRNAs defined in our study (Figs 2H and 3) with the same tsRNA in the Donkin and colleagues study revealed similar expression levels across stud- ies (Fig 4C). Examining tRNA profiles from both studies side by side revealed further similari- ties, both in the relative abundance of different tsRNAs and nutrient sensitive regions (S4 and S5 Figs). Interestingly, in line with the anabolic effect of the high-sugar diet in young and healthy men, sperm from clinically obese men showed opposite responses in tsRNA changes (Fig 4D). Where high-sugar was associated with an increase in sugar-sensitive tsRNA, obesity was associated with a decrease. Notably, this inverse relationship was also observed in the sugar-sensitive 12S and 16S rsRNA (S2D/S2E Fig). To further elaborate on this, we compared our results with data from a recent study by Hua and colleagues that investigated sncRNA from sperm of Chinese men that later generated Fig 4. Sperm from obese men display changes in tsRNAs. (A) Previously published data by Donkin and colleagues on sperm small RNA from obese and lean men (SRA project: SRP065418) [25] was reanalyzed using our analytical workflow. (B) Boxplot show mean, and individual expression, of the different tsRNA types (grey dots lean individuals, black dots obese individuals). Error bars indicate ± SEM. (C) Shows a significant relationship between average tsRNA expression across the current (Sugar/Healthy) and the obesity (Obesity/Lean) studies. Each colored large dot represents individual tsRNAs from significantly changed, sugar-sensitive tRNA isodecoders in Fig 2H. Grey small dots represent other tsRNA. (D) Same tsRNAs as in panel C but plotted as the differences within each of the two studies, comparing Sugar versus Healthy diets, and Obese versus Lean men, respectively. Percentages represent the proportion of tsRNA found in each quadrant of the plot; not bracketed = diet sensitive; in brackets = all analyzed tsRNA. RPM, reads per million; SRA, Sequence Read Archive; tsRNA, tRNA-derived small RNA. https://doi.org/10.1371/journal.pbio.3000559.g004 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 7 / 25 Human sperm displays rapid responses to diet high- or low-quality embryos after in vitro fertilization [34]. While we successfully identified many of the sugar-sensitive tsRNA and rsRNA, we only observed weak trends with increased expression of mitochondrial rsRNA and tsRNA associated with higher embryo quality (S2 and S5 Figs). The Hua and colleagues dataset was, however, generally depleted in rsRNA and heavily enriched in 50 tsRNA compared to the other datasets (S6 Fig). Therefore, the effect on sugar-sensitive i-tsRNA and rsRNA may have suffered from low coverage or overlapping deg- radation products of more abundant fragments (e.g., LysCTT in S4 Fig). Together, the cross-dataset analysis in combination with our qPCR verification (S3 Fig) provide solid independent evidence of the existence of the novel sugar-sensitive tsRNA identi- fied in this study. Moreover, the inverse correlation with tsRNA in sperm from obese men sug- gests that they might be involved in a clinical condition. Sugar-sensitive nuclear i-tsRNAs are cleaved in the T-loop Next, we focused on the significantly changed nuclear isodecoders from Fig 2H. Aligning reads to the best match of LysCTT, ArgCCG, ArgCCT, and LeuCAA showed that they had very similar coverage profiles (Fig 5A–5D, top rows), despite unique sequences and originating from different chromosomes. This was in stark contrast to the complexity of tRNA fragments originating from significantly changed mitochondrial isodecoders (S7 Fig). More specifically, all nuclear sugar-sensitive isodecoders had 30 -end positions close to or within the T-loop. To investigate their exact cleavage sites, we mapped the first and last nucleotide for each fragment (Fig 5A–5D, middle and bottom rows). This mapping revealed that the sugar-sensitive frag- ments for LysCTT had a highly specific 30 cut-site within the T-loop and a 50 cut-site between the D-loop and anticodon-loop in the anticodon arm, generating a 30-nt-long fragment (Fig 5E). ArgCCG, ArgCCT, and LeuCAA fragments were smaller, 16–22 nt in length, all starting in various positions after the anticodon but ending in, or very close to, the T-loop (Fig 5F– 5H). All 4 significant sugar-sensitive nuclear isodecoders had a TTCGA sequence in their T-loop (Fig 5I). This is a common sequence found in 48% of all T-loops from tRNA-related sequences, of which less than 1% have mitochondrial origin (compared with 6% in non–TTCGA-contain- ing tRNAs). To investigate whether sugar-sensitive fragments were enriched in TTCGA-T- loops, we mapped the distance between the nucleotide at the 30 -end of each tsRNA and the 50 position of the T-loop (Fig 5J, top panel) (S4 Table). As expected, this resulted in two major peaks, corresponding to the highly expressed 50 -tsRNAs and 50 -halves of GlyCCC, GlyGCC, GluCTC, and GluTTC, whereas the moderately expressed sugar-sensitive T-loop tsRNA showed a smaller peak (Fig 5J, top panel, insert). Plotting the significance value for sugar-diet– induced changes revealed two things. First, the T-loop was indeed a hotspot for significantly changed nuclear tsRNAs in tRNAs with a TTCGA motif (Fig 5J, middle panel, filled blue cir- cles p � 0.05, open blue circles p > 0.05). The few nuclear tsRNAs without a TTCGA motif did not show this accumulation of significant tsRNAs (Fig 5J, lower panel, filled blue circles p � 0.05, open blue circles p > 0.05). Second, tsRNAs from mitochondrial tRNA showed a more complex pattern of sugar-sensitive cut-sites (Fig 5J, middle and lower panel, filled red circles p < 0.05, open red circles p > 0.05). Together, this suggests that the mechanism under- lying the discrimination of sugar-sensitive nuclear i-tsRNA, which was identified by our clus- ter analysis, likely involves selective T-loop cleavage in a predefined subtype of tsRNA. We name this nuclear tsRNA subtype—defined by their TTCGA-T-loop cleavage—nuclear inter- nal T-loop tsRNA (nitRNA). Notably, our T-loop mapping also revealed sugar-sensitive nitRNA in GluCTC that previously was hidden in abundant diet-insensitive 50 tsRNA (S8 Fig, S4 Table). PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 8 / 25 Human sperm displays rapid responses to diet Fig 5. T-loop cleavage generates sugar-sensitive nitRNAs. (A–D) Nucleotide coverage and cleavage site analysis. (E–H) Graphical representation of tsRNAs generated in response to a high-sugar diet. (I) Shared T-loop sequence (TCCGA) across sugar-sensitive nuclear tsRNAs. (J) Positional analysis of 30 cleavage sites including all tsRNAs in the study. Top panel: Sugar (dark grey) versus Healthy (light grey) diet. Middle and bottom panels: significant tsRNAs (filled colored dots; p < 0.05), nuclear tsRNAs (blue), and mitochondrial tsRNAs (red). Data are available in S2 Data and S4 Table. nitRNA, nuclear internal T-loop tsRNA; RPM, reads per million; tsRNA, tRNA-derived small RNA. https://doi.org/10.1371/journal.pbio.3000559.g005 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 9 / 25 Human sperm displays rapid responses to diet Diet-sensitive tsRNA correlates with sperm motility Because sperm motility is one of the best indicators of male subfertility [35], is reduced in obese men [9,36], and was stabilized at high levels during the course of our 2-week diet inter- vention (Fig 1E), we tested whether changes in sperm motility were associated with the sugar- sensitive tsRNA. Since the largest recovery of sperm motility was observed between the start point and after the high-sugar diet, we focused on the differences between these two time points (Start versus Sugar). By doing so, we also dissociated the original analysis between Healthy and Sugar, enabling possible dependencies on starting conditions to emerge. First focusing on nuclear tsRNA, we found that sugar-sensitive nitRNA was not up-regu- lated in response to a healthy diet but specifically to a high-sugar diet (Fig 6A, dark green cir- cles). This was not a general response of nuclear tsRNA because the mean of all other nuclear tsRNA was unaffected by the intervention (Fig 6A, light green circles). Furthermore, on an Fig 6. Sperm motility is positively associated with parallel changes in tsRNA. Nuclear and MT tsRNA—either sugar-sensitive (= significantly up-regulated in Sugar versus Healthy) or other (= not significantly up-regulated tsRNA) —were reevaluated in relation to Start point values. (A) Shows the changes in nuclear sugar-sensitive nitRNA (dark points) and other nuclear tsRNA (light points) over the whole diet intervention. Note that sugar-sensitive nitRNA specifically responded to Sugar. (B) Changes in sperm motility between Sugar versus Start diets were associated with sugar-sensitive nitRNA (dark points), but not other nuclear tsRNA (light points). (C) No association between changes in nitRNA and other nuclear tsRNA. (D) Changes in sugar-sensitive MT tsRNA (dark points) and other MT tsRNA (light points) in relation to Start point values. Note that all MT tsRNA progressively increased over the diet intervention, but a boost was seen in sugar-sensitive after Sugar. (E) Changes in both sugar-sensitive MT tsRNA (dark points) and other MT tsRNA (light points) were associated with changes in sperm motility. (F) Changes in sugar- sensitive MT tsRNA and other MT tsRNA were strongly correlated. ���� p < 0.0001, �� p < 0.01, � p < 0.05, # p < 0.1. Data are available in S2 Table (sperm motility) and S2 Data (tsRNA). Healthy, samples taken after healthy diet; MT, mitochondrial; nitRNA, nuclear internal T-loop tsRNA; n.s., not significant; Start, start point samples; Sugar, samples taken after high-sugar diet; tsRNA, tRNA-derived small RNA. https://doi.org/10.1371/journal.pbio.3000559.g006 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 10 / 25 Human sperm displays rapid responses to diet individual level, changes in sugar-sensitive nitRNA were positively associated with changes in sperm motility, while other nuclear tsRNA was not (Fig 6B). Neither was there an association between nitRNA and other nuclear tsRNA (Fig 6C). Contrary to nuclear tsRNA, mitochondrial sugar-sensitive and all other mitochondrial tsRNA both showed an increase across the 2-step diet intervention (Fig 6D). While other tsRNA showed a steady increase, the sugar-sensitive tsRNA experienced an acceleration after the sugar was introduced (dark red circles Fig 6D). Accordingly, mitochondrial tsRNA initially identified as sugar-sensitive, as well as other mitochondrial tsRNA, both correlated positively with sperm motility (Fig 6E). The mean changes of sugar-sensitive and other tsRNA also strongly correlated with each other (Fig 6F). This indicates that sperm motility depends on a common mechanism involving all mitochondrial tsRNA. Because sperm motility and mito- chondrial tsRNA increased already the first week (Fig 1E; Healthy), the healthy/baseline diet was at least partly responsible for the positive effects on sperm quality. Discussion Here we show that human sperm are sensitive to nutritional flux, both in respect to sperm motility and the sncRNA pool. Such an acute responsiveness agrees with our earlier study in Drosophila, in which we showed that just 2 days of dietary intervention in male flies before mating was sufficient to transmit a signal through the sperm to induce metabolic reprogram- ming in the next generation [19]. In the current study, we show that specific mitochondrial and nuclear tsRNAs in human sperm are independently up-regulated following a similar short-term diet intervention. The increase of these tsRNA was positively associated with sperm motility. Moreover, in nuclear tsRNAs, we identified sugar-sensitive cleavage in the T-loop of full-length nuclear tRNA, generating a short internal tsRNA, which we named nitRNA. Biogenesis of sperm nuclear tsRNA Pathways that generate tsRNA from tRNA is only partly understood. Best understood is the stress-induced cleavage of tRNA by angiogenin that cuts in the codon-loop giving rise to 2 halves [37,38]. We find, as reported earlier in mouse [26], that the most abundant tsRNA in sperm are 50 -halves. We do not, however, find evidence that tsRNA with intact 50 ends are responding rapidly to diet. Using novel approaches to map internal tRNA fragments, we instead detected another class of tsRNAs, i-tsRNA, that is not as highly expressed as many 50 -tsRNA, but compared to 30 - tsRNA still is expressed at intermediate levels (Fig 2F). In mice, it has been shown that 50 - tsRNA is up-regulated in sperm as a response to a chronic low-protein diet [26], a chronic high-fat diet [24], or a maternal high-fat diet [28]. Considering the role of 50 -tsRNA in inhibit- ing translation [29,37], up-regulation of 50 -tsRNA could be a natural response for reducing protein synthesis under amino acid deprivation. Our acute high-sugar intervention results in a different metabolic situation. The participants are first well-nourished and then receive a high- sugar dose on top of that. This could explain why the tsRNAs that we identify are different from studies on low protein and high fat. Given such dependencies on metabolic backgrounds, this would imply the possibility that different diets may have different consequences on the sperm itself, and maybe even on the developing zygote. It must, however, be noticed that most of the earlier studies have neglected the internal—i-tsRNA—fragments. Thus, reanalyzing the data may reveal more commonalities in sugar-sensitive tsRNA pathways. The sugar-sensitive nitRNAs identified in this study all had GTTCGA motifs in the T-loop. This is the exact motif for Pseudouridine Synthase 7 (PUS7), an enzyme that catalyzes pseu- douridylation on uridines. Intriguingly, several of the tRNA isodecoders that we have defined PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 11 / 25 Human sperm displays rapid responses to diet as sugar-sensitive in the present study are targets of PUS7 [39]. Even though there are remark- able similarities between the sugar-sensitive tsRNA found in this study and the tRNA targets for PUS7, more work is needed to determine whether there is a role for PUS7 in diet-induced nitRNA generation in sperm. T-loop cleavage should in addition to the identified nitRNA generate short 30 -tsRNAs (S9 Fig). Such short 30 -tsRNAs were rare in our data but have been described [40]. In Tetrahy- mena, there is a starvation-induced longer 30 -tsRNA that is processed by the piwi pathway to generate a CCA-30 -tsRNA [41]. The “left-over” of the longer 30 -tsRNA is very similar to the nitRNA that we have found to be increased in sperm in response to a high-sugar diet. One explanation for the lack of short 30 -tsRNA in our study is that these fragments may contain posttranslational modifications that are incompatible with our library preparation protocols. This should be addressed by future studies. Paralleled shift in sperm motility and tsRNA Sperm motility increased over the course of the study and was prominent already after the first week of a healthy diet. While we cannot dissociate the effect of the underlying healthy diet from high-sugar intake, it is possible that the increase of sperm motility was a direct conse- quence of the healthy diet, which may have persisted into the second week. Glucose has, however, multiple roles in mature spermatozoa [42] and is known to rapidly affect sperm motility in vitro [43,44]. This is likely because mature human spermatozoa use glucose and fructose as primary source of ATP. As shown in boar semen, mitochondrial tran- scription, unlike nuclear transcription, appears fully active and is dependent on ATP [45]. Therefore, it is possible that what we see as an increase of mitochondrial tsRNA is a result of more full-length mitochondrial tRNA caused by sugar-dependent gene transcription. Such claims must of course be further investigated. Nevertheless, while we did not measure glucose levels in seminal fluid directly, we did not observe changes in blood glucose levels during the experiment (S2 Table), which makes direct sperm glucose sensing an unlikely explanation for our results. No changes in blood glucose, as well as increased triglyceride levels (S2 Table), instead suggest that participants expressed a healthy sugar metabolism and, under the influ- ence of hormonal responses, quickly converted blood sugar to fatty acids. Mature spermatozoa are known to carry leptin receptors [46] and have insulin sensing capabilities [47], which in vitro positively affect sperm motility [48]. If such hormonal pathways would be connected to an intracellular cascade that activates PUS7-like pseudouridinylation (see discussion in “Bio- genesis of sperm nuclear tsRNA”) and subsequent T-loop cleavage of the preexisting pool of sperm tRNA, this would be a strong candidate for explaining at least some of our results. Given that spermatogenesis takes approximately 70 days in humans [49]—where at least nuclear gene transcription is strongly repressed during the later stages (see [50] for a discus- sion on this)—it may seem unlikely that rapid changes in the sncRNA pool are mediated by sperm endogenous gene transcription. Thus, rapid changes are more likely explained by either tsRNA processing from a preexisting pool of tRNA or transfer of tsRNA from somatic cells with intact gene transcription (Fig 7). Nutrient sensing may therefore work either via direct or indirect sensing mechanisms, such that the sperm itself or somatic cells sense the dietary flux and execute a response. Recent findings in mice suggest that somatic cells in the male reproductive tract is capable of transferring sncRNA across the seminal lumen and into maturing spermatozoa [26,27,51– 53]. This soma-to-germline transfer of RNA is a potential route for passing information from one generation to the next and suggests mobile RNA as a molecular mechanism for intergener- ational epigenetic inheritance [54,55]. The transfer of RNA is thought to be mediated by PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 12 / 25 Human sperm displays rapid responses to diet Fig 7. Alternative hypotheses for rapid responses to diet in human sperm. Since endogenous nuclear gene transcription in late-stage spermatozoa is strongly repressed, rapid responses are likely dependent on the activation of latent factors already available within the sperm or on the transfer of critical factors from surrounding somatic cells. Possible mechanisms for latent factor activation involve direct sperm sensing of dietary nutrients present in seminal fluid or intercellular signaling by receptor-ligand binding. Exosomes, which are small extracellular vesicles, known to transfer sncRNA between cells, are candidates for soma-to-sperm transfer of sncRNA such as tsRNA, but also other factors affecting sperm motility. In humans, epididymal principal cells and prostate acinar cells in the male reproductive tract are known to release such exosomes into seminal fluid. Thus, exosome transfer is the only known mechanism that may increase nuclear sncRNA in late-stage sperm by de novo transcription. Transcription of mitochondrial sncRNA in mature sperm is less well understood. sncRNA, small non-coding RNA; tsRNA, tRNA- derived small RNA. https://doi.org/10.1371/journal.pbio.3000559.g007 exosomes, which are small extracellular vesicles known to carry a range of biologically active molecules—including sncRNA—and are abundant in human seminal fluid [56,57]. In humans there are two types of exosomes described in the male reproductive tract: epididymosomes, originating from epididymal principal cells, and prostatasomes from acinar cells in the pros- tate. Small RNA-seq of prostatasome from human seminal fluid has been shown to contain sncRNAs, including tsRNA [57], but it is not yet known whether they can deliver their cargo to mature sperm. Nevertheless, prostatasomes can increase sperm motility [58]. Thus, our findings of a simultaneous increase of sperm motility and tsRNA in response to a short dietary intervention is in line with an exosomal soma-to-germline transferring pathway. Is there a role for tsRNA in metabolic intergenerational effects in humans? In mice, it has been shown that 50 -GlyGCC, which is abundant in sperm and affected by a low-protein diet, represses genes necessary for the proliferation of murine endogenous retro- viruses in embryos and in embryonic stem cells [26]. Moreover, short CCA-30 -tsRNAs have been shown to inhibit this type of retrotransposons in mouse preimplantation stem cells [59]. It has, therefore, been proposed that tsRNA protects against reactivation of transposable ele- ments during the reprogramming to a pluripotent stage [59]. There are multiple lines of evi- dence connecting the regulation of transposable elements to metabolic phenotypes. First (and still one of the best examples of epigenetic inheritance of obesity) is the transgenera- tional control of a retrotransposon upstream of the agouti gene in mice [60]. Moreover, SETDB1 and TRIM28 (also known as KAP1), which both repress retrotransposons, also modulate obesity in mice [61,62]. Thus, it is possible that sperm tsRNAs—via direct control of retrotransposons, or genes harboring regulatory elements borrowed from retrotranspo- sons—set long-term metabolic programs in the embryo that later define offspring obesity risks. Considering the accumulating evidence of sncRNA being a mobile source of cell-to-cell communication, it is an intriguing idea that this communication is effective also from sperm to egg. This is robustly supported by studies in mice [24,26,28], but due to ethical constraint, PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 13 / 25 Human sperm displays rapid responses to diet such studies are difficult to perform on humans. Even though the recent study on embryo quality by Hua and colleagues [34] supports this idea, conclusive evidence that the human egg cell is responding to changes in the pool of sperm sncRNA, or what has been termed the sncRNA code [63], is missing. We have, however, shown that human sperm has the plasticity to reconfigure the sperm sncRNA code in response to rapid environmental changes, which in other species has served as a message to the next generation. Most importantly, the paralleled response of sperm motility and shifts in the sncRNA code hints that there might be a shared etiology between male fertility and intergenerational metabolic responses. We conclude that human sperm is acutely sensitive to dietary shifts and propose that this sensitivity involves an interplay between the sncRNA code and sperm function. This is likely driven by two independent pathways, divided by cellular compartmentation (nuclear/mito- chondrial). Further exploration of these pathways may not only be critical in understanding the global decline in human sperm function but may provide a possible mechanism for rapid intergenerational metabolic responses so far only described in animals. Materials and methods Ethics statement The study was approved in accordance with the Declaration of Helsinki by the regional ethical board at Linköping University, Sweden (permit number: 2016/183-31). We obtained written informed consent from all participants. Study design and participants The diet intervention was performed during a 2-week period. It included 15 voluntary men recruited by advertising at the University of Linköping, Sweden. The inclusion criteria were being between 20 and 30 years old, not having obesity (i.e., BMI < 30.0 kg/m2), being a non- smoker, and being an omnivore. For the 2 weeks of diet intervention, the recruited partici- pants committed to refraining from alcohol and were asked to keep their activity levels constant. Calculation of TEE Before starting the intervention, the participants’ physical activity level (PAL) was estimated using a questionnaire comprising questions regarding their PAL at work and at home/leisure time [64]. BodPod (COSMED USA, Concord, CA) was used to measure fat and lean mass. We also measured weight and height of the participants, by which the participants’ basal metabolic rate (BMR) was predicted according to age [65]. The participants’ TEE was then calculated as TEE = BMR × PAL [32]. The estimated TEE for each participant was assumed to correspond to their individual daily energy requirement (i.e., the energy intake required to maintain their current body weight) (S1 Table). Diet intervention During the first week of the diet intervention, participants (n = 15) received a standardized healthy diet with an energy intake corresponding to their estimated TEE. The diet was designed to meet the nutritional recommendations of the Nordic countries [32] and assumed the following energy distribution over meals: breakfast 25%, lunch 30%, dinner 30%, and snacks 15%. All meals were provided by the research team, and the participants were instructed to only drink water during the first week of the study. For breakfast, they were pro- vided with natural yoghurt (3% fat), breakfast cereals (whole grain), natural hazelnuts, banana, PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 14 / 25 Human sperm displays rapid responses to diet crisp bread, butter (60% fat), hard cheese (28% fat), egg, and pepper. For snacks, the partici- pants received banana, apple, cherry tomatoes, baby carrots, and natural almonds. Lunch and dinner were provided by the university restaurant in close collaboration with the nutritionists of the research team and followed the same careful standardization as all other meals. During the second week, the participants received the same standardized healthy diet as described above. In addition to this diet, the participants also consumed candy (not including chocolate and licorice) and sweetened beverages corresponding to 50% of their estimated TEE. The balance between candy and sweetened beverages was adjusted according to their own preference to optimize compliance. The intake of candy and sweetened beverages together with the standardized healthy diet resulted in an energy intake that corresponded to 150% of their estimated TEE. Semen collection Semen samples were obtained from each person within the study group (n = 15) at three time points at Reproductive Medicine Center, University Hospital of Linköping between November 2016 and October 2017. Samples were produced by masturbation after 2 to 3 days of sexual abstinence, collected in sterile 50 ml non-spermiotoxic polypropylene tubes and allowed to liq- uefy at room temperature. After liquefaction of semen, standard semen parameters (volume, density, motility, white blood cells) were obtained according to World Health Organization criteria [66]. Preparation of motile spermatozoa and further processing Motile spermatozoa were prepared by using a discontinuous (1.5 ml 80%/1.5 ml 40%) Pure- Sperm (Nidacon Int, Gothenburg, Sweden) gradient. Neat semen (0.5–2.0 ml) was layered on the top of the gradient and centrifuged at 300g for 20 minutes, followed by resuspension of the pellet in equilibrated sperm preparation media (PureSperm Wash; Nidacon). The sperm sus- pension was thereafter centrifuged at 500g for 10 minutes and resuspended and diluted to appropriate concentration/volume motile sperm (using a Makler counting chamber; Cellvi- sion, Heerhugowaard, the Netherlands) in equilibrated PureSperm Wash. The proportion of motile spermatozoa after preparation was always >97%. Aliquots of prepared motile sperm (0.03–0.10 ml; total motile sperm count per ejaculate 0.35 to 21 million) were thereafter trans- ferred to 1.5 ml PCR-clean microtubes followed by immediate freezing in liquid nitrogen (−196 ˚C). The frozen sperm suspensions were stored at −80 ˚C until later extraction of sperm RNA. RNA extraction RNA extraction was done using miRNeasy Micro kit (Qiagen, Venlo, the Netherlands) and performed according to the manufacturer instructions with minor adjustments. Great care was taken so that the sperm samples never thawed before homogenization. To the frozen sam- ples, 0.15 g of frozen 0.2 mm steel beads (SSB02-RNA NextAdvance, Troy, NY) were added followed by addition of prechilled Qiazol (Qiagen). Samples were quickly moved to a Tissue Lyser LT (Qiagen) and homogenized at 30 oscillations/second for 5 minutes, followed by heat- ing to 37 ˚C and then homogenization for another 3 minutes [67]. Chloroform was added, and phase separation was performed by centrifugation at 12,000g. Upper phases were collected and applied on RNase MinElute Spin Columns provided with the kit. Columns were then repeat- edly washed, before RNA was eluted in 14 μl of water and stored at −70 ˚C until library preparation. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 15 / 25 Human sperm displays rapid responses to diet Library preparation Library preparation was done with NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA) according to the manufacturer instructions with the following minor customizations. Samples were standardized by sperm input counts, and primers in the kit were diluted 1:7 accordingly. Amplified libraries were cleaned using Agencourt AMPure XP (Beckman Coulter, Brea, CA) and size selected for 130 to 165 nt fragments on a pre-casted 6% polyacrylamide Novex TBE gel (Invitrogen, Waltham, MA). Gel extraction was done using Gel breaker tubes (IST Engineering, Milpitas, CA) in the buffer provided in the NEBNext kit. Disintegrated gels were incubated at 37 ˚C for 1 hour on a shaker, quickly frozen for 15 min- utes at −80 ˚C, followed by another incubation for 1 hour. Any remaining gel debris was removed by Spin-X 0.45 μm centrifuge tubes (Corning Inc., Corning, NY) as recommended by the NEBnext protocol. The libraries were precipitated overnight at −80 ˚C by adding 1 μl of GlycoBlue (Invitrogen) as co-precipitant, 0.1 times the volume of Acetate 3M (pH 5.5), and 3 times the volume of 100% ethanol. Library concentrations were estimated using QuantiFluor ONE ds DNAsystem on a Quantus fluorometer (Promega, Madison, WI). Pooled libraries were sequenced on NextSeq 500 with NextSeq 500/550 High Output Kit version 2, 75 cycles (Illumina, San Diego, CA). All pooled libraries passed Illumina’s default quality control. Preprocessing of sequenced reads S1 Text contains an R script with instructions and functions on how to automatically import data from S1–S4 Tables and S1 and S2 Data and regenerate some of the results presented in the main article. Updates on the script will be posted on https://github.com/Danis102. Cutadapt version 1.9.1 [68] was used to trim any remains of adaptor sequence (AGATCG GAAGAGCACACGTCTGAACTCCAGTCA) from sequenced reads. Only trimmed reads between 15–45 nucleotides, containing adaptor sequence, and with 80% of the nucleotides showing Illumina quality scores (Q-scores) >20 were retained. Average depth was 30.05 ± 1.14M reads per sample (min = 17.23M) in which at least 81.54% passed our initial fil- ters. For initial analysis, trimmed reads were mapped to small RNAs using Sports 1.0, a perl- based analytical workflow well suited for many types of small RNA, including rsRNA [69]. We used the default settings and database files for the human genome that are available on the Sports github (https://github.com/junchaoshi/sports1.0; accessed June 2019). This included the hg38 UCSC genome files, miRNA from miRbase 21, rRNA from NCBI/Nucleutide, tRNA from GtRNAdb, piRNA from pirBase and piRNAbank, other ncRNA from ensembl (release- 89), and rfam 12.3. Averages summarized over biotypes (rsRNA, tsRNA, miRNA, etc.) were based on the default annotations in Sports result output files. To avoid too much noise, a non- conservative coverage filter was applied that discarded fragments with < 0.01 reads per million (RPM) in 33% of the samples (3 samples per participant). Sports output data are available in S1 Data. tRNA specific mapping and analysis using MINTmap Since Sports currently do not support advanced analysis of mitochondrial tRNA, for more spe- cific tRNA analysis, MINTmap version 1.0 [33] was used instead. Adaptor-trimmed and qual- ity filtered reads were mapped against the MINTbase tRNA database containing 640 full- length tRNA sequences. We used default settings for the GRCh37/hg19 human genome. The analysis included tsRNAs that exclusively mapped to tRNA sequences, tsRNAs that ambigu- ously mapped tRNA sequences with possible additional alignments to genomic sequences out- side tRNA space, and tRNA lookalike sequences. Classifications together with MINTmap PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 16 / 25 Human sperm displays rapid responses to diet outputs, normalized to RPM in relation to the total reads in the original fastq file, are presented in S2 Data. Annotations for isoacceptor/decoders (e.g., LysCTT, ArgCGG), tsRNA subtype (50 -half, 50 - tsRNA, i-tsRNA, 30 -tsRNA, 30 -half), and full tRNA sequences were obtained from the MIN- Tmap software files, while tRNA loop annotation was obtained from the MINTbase webpage (https://cm.jefferson.edu/MINTbase/; accessed 2018-11-29). MINTbase defines tsRNA sub- types as follows: 50 -tsRNAs and 50 -halves align with their first position in the first position of the mature tRNA, whereas 30 -tsRNAs and 30 -halves have the full-length tRNA termination sequence—CCA—in their 30 ends. The 50 - and 3’-halves are exact fragments, leaving the 50 - and 30 -tsRNAs to be everything else, either shorter or longer then the halves. The fifth subtype, i-tsRNAs, are any fragment that neither starts nor ends in the initial or terminal part of the full-length tRNA. RPM values were analyzed using different packages in R version 3.5.1 and Bioconductor version 3.7. If not otherwise stated, we applied a coverage filter discarding fragments with < 1 RPM in 33% of the samples and a size filter retaining fragments between 16 and 45 nucleo- tides. In total, 1,725 unique fragments were retained, which are 3.75% of all sequences detected by MINTmap. These numbers were expected because most fragments were only detected at low coverage in one or a few samples and because detection above 45 nucleotides was biased by gel size selection procedures. Since full-length tRNAs often occur in several related copies, containing sequences that are semiconserved across these copies, some fragments will map to multiple full-length tRNAs. Multimapping issues is therefore reported in S4 Table and S1/S2 Data. To be able to visualize our results, we consistently report the best aligned full-length tRNA showing the highest RPM coverage. It must be emphasized, though, that some of the fragments may have originated from other full-length tRNAs that shared sequence with the best aligned/covered full-length tRNA. To map each fragment to single nucleotide resolution in best aligned/covered full-length tRNAs, we used the vmatchPattern function in Biostrings version 2.48 [70], while tRNA cover- age was calculated on discretized RPM values using the coverage function in GenomicRanges 1.32.6 [71]. Since MINTmap adds undetermined nucleotides (NNN) to the 30 and 50 ends, this was also added to our sequence alignment and coverage analysis. For T-loop analysis and plot- ting, stringr version 1.3.1 [72] was used to standardize the T-loop starting position to the first occurrence of the conserved TTC or ATC sequences (accounting for 80.1% and 6.1% of all 640 tRNAs, respectively). A similar approach was used to locate TTCGA motif in the T-loop. Start- ing position for T-loops without TTC or ATC was set to the first 50 nucleotide in the T-loop. The distance between the fragment 30 end and T-loop 50 start was then calculated. To deal with multimapping issues, fragments were assigned the most common distance across multimap- ping full-length tRNAs. Graphs were generated using ggplot2 version 3.0 [73]. tsRNA analysis on external datasets For comparison to relevant previously published data on human sperm small RNA, we down- loaded raw fastq files from a cohort of lean (n = 13) and obese (n = 10) adult men, published by Donkin and colleagues and available at NCBI’s Sequence Read Archive (SRA) project (SRP065418; Gene Expression Omnibus [GEO] access: GSE74426) [25], using SRA toolkit ver- sion 2.9.2 [74]. Similarly, data from a study published by Hua and colleagues investigating sperm RNA in relation to high (n = 23) and low (n = 64) embryo quality following in vitro fer- tilization (SRA project: SRP132262; GEO access: GSE110190) [34] was downloaded. Raw reads were trimmed, quality filtered, and imported into MINTmap as described earlier. Our PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 17 / 25 Human sperm displays rapid responses to diet quality check detected a severe outlier among the lean samples of the Donkin and colleagues dataset, which was removed from further analysis (final dataset: lean n = 12; obese n = 10). Since adaptor trimmed reads from the Donkin and colleagues dataset had a max read length of 32 nucleotides, the Swedish dataset only included tsRNAs with �32 nucleotides when analyzed across studies. tsRNA validation using qPCR Quantitation of tsRNA using stem-loop retro-transcription (RT) has been described elsewhere [39]. In brief, 20 ng of sperm total RNA was incubated at 65 ˚C for 5 minutes with 0.5 μl 10 mM dNTPs and 1 μl tsRNA specific stem-loop RT primer (LysCTT: GTCGTATCCAGTGCA GGGTCCGAGGTATTCGCACTGGATACGACAACCCA; MT_SerTGA: GTCGTATCCAG TGCAGGGTCCGAGGTATTCGCACTGGATACGACCAACCC). Samples were then imme- diately placed on ice for 2 minutes, followed by the addition of 6.45 μl of RT mix, 4 μl 5X First Strand buffer, 2 μl 0.1M DTT, 0.2 μl RNase OUT, and 0.25 μl Superscript III (Thermo Scien- tific, Waltham, MA). RT was performed at 16 ˚C for 30 minutes, 60 cycles at 30 ˚C for 30 sec- onds, 42 ˚C for 30 seconds, and 50 ˚C for 1 second, and ended with enzyme inactivation at 85 ˚C for 5 minutes. Resulting cDNA was diluted 1:5, before qPCR was performed using SsoAd- vanced Universal SYBR Green Supermix (BioRad, Hercules, CA), with tsRNA specific forward primer (LysCTT: TGGGACTCTTAATCCCAGGG; MT_SerTGA: AAAGTCATGGAGGCCA TGGG) and a universal reverse primer on the stem loop adapter (GTGCAGGGTCCGAGGT). Product specificity was validated by agarose gel electrophoresis and by absence of signal in no- template and no-RT controls. Statistics All tsRNA/rsRNA sequencing analysis was done on discretized RPM values, imported into a repeated measure mixed linear model with negative binomial distribution using the lme4 package version 1.1.18.1 [75]. This model was motivated by a paired sample design, and because RPM values are based on read counts, which have previously shown best coherence with a negative binomial rather than a Gaussian or a Poisson distribution [76]. Since there is currently no method to model the statistical dependency between closely related tsRNAs that may or may not originate from the same tRNA (see subheading “tRNA specific mapping and analysis using MINTmap”), we did not adjust our p-values for multiple testing. Without know- ing the dependency between tsRNAs, there is a severe risk for committing type II (false nega- tives) errors using any existing approach to correct for multiple testing in tsRNA-seq analysis. Cluster analysis and visualization were done using hierarchal clustering on Euclidian distances from center scaled data using ape 5.2 [77]. For qPCR validation, paired and sided (greater expression in Sugar than Healthy) Wilcoxon signed-rank tests were used. Supporting information S1 Fig. Size distribution and first nucleotide bias of sperm small RNA. Histograms for all ejaculates from all participants (n = 15 participants ×3 repeated ejaculates). Colors for first N; Red = A, Green = C, Blue = G, Purple = T). (EPS) S2 Fig. Analysis of rsRNA using Sports 1.0. (A) Participant mean expression of rsRNA summed over full-length nuclear (5S, 5.8S, 18S, 28S) and mitochondrial (12S, 16S) rRNA sub- units. 45S is the pre-rRNA transcription unit, with rsRNA mapping to 5.8S, 18S, and 28S sub- units excluded, leaving only the 2 spacers. (B) Fold change differences per rsRNA of sugar PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 18 / 25 Human sperm displays rapid responses to diet versus healthy diet. Circle diagrams show the percentages of up-/down-regulated fragments. (C) Fold change differences of each participant. Only mitochondrial rRNA subunits are up- regulated. (D) Coverage over the different rRNA subunits using data from the current study (left column), and data from 2 contrast studies targeting sperm small RNA differences of obese versus lean men (middle column [25]) and men generating high- or low-quality in vitro fertil- ized embryos (right column [34]). Arrows show highest peak of each subunit in the current study. (E) Differences in sperm rsRNA subunit expression between lean (light) and obese (dark) men. Mitochondrial subunits are down-regulated. (F) As (E) but between high- (light) and low- (dark) quality embryos. The 45S spacers and possibly 12S (see D) are up-regulated in high quality. � p < 0.05 and �� p < 0.01 negative binomial generalized linear (contrast stud- ies) or mixed linear (current study) models. Data are available in S1 Data, SRA project: SRP065418, and SRA project: SRP132262. (EPS) S3 Fig. Validation of tsRNA fragments using qPCR. Two diet-sensitive tsRNAs—the nuclear LysCTT i-tsRNA (nitRNA) and mitochondrial SerTGA 50 -tsRNA—were chosen for qPCR val- idation. (A) Mapping of the tsRNA (bold) over the full-length tRNA. (B) Both assays success- fully amplified in hESCs with near to exponential efficiencies. (C) Sequencing and qPCR results correlated well across all participants (n = 30; 15 Healthy, 15 Sugar). Note that a high Ct value means lower expression. (D) Both sperm and hESCs generated unique fragments in the expected sizes after qPCR. (E) A sided Wilcoxon signed-rank test also confirmed that the expression was greater in Sugar than in Healthy. Together, the qPCR experiment confirmed the presence of the tsRNA in Sperm, as well as in hESC, and validated the effect of the diet intervention. Data are available in S3 Data. hESC, human embryonic stem cell; MT, mitochon- drial. (EPS) S4 Fig. Reproducible tsRNA coverage on diet-sensitive nuclear tsRNAs across independent studies. Graphs show side-by-side comparisons between the present study (left column) and the Donkin and colleagues obesity study [25] (middle column) and the Hua and colleagues embryo quality study [34] (right column) on the nucleotide coverage over full-length tRNA sequences. Left column; Red lines = Sugar diet, Blue lines = Healthy diet. Middle column; Red lines = Obese men, Blue lines = Lean men, Right column; Red lines = High quality embryos, Blue lines = Low quality embryos. Data are available in S2 Data, SRA project: SRP065418, and SRA project: SRP132262. (EPS) S5 Fig. Reproducible tsRNA coverage but inversed effects on diet-sensitive mitochondrial tRNAs across independent studies. Graphs show side-by-side comparisons between the pres- ent study (left column) and the Donkin and colleagues obesity study [25] (middle column) and the Hua and colleagues embryo quality study [34] (right column) on the nucleotide cover- age over full-length tRNA sequences. Left column; Red lines = Sugar diet, Blue lines = Healthy diet. Middle column; Red lines = Obesity, Blue lines = Lean, Right column; Red lines = High quality embryos, Blue lines = Low quality embryos. Data are available in S2 Data, SRA project: SRP065418, and SRA project: SRP132262. (EPS) S6 Fig. Mean composition of different small RNA subtypes across studies. Current study was compared to two other studies, Donkin and colleagues [25], and Hua and colleagues [34]. Diagrams were constructed using the identical default workflow in Sports 1.0, while maintain- ing only fragments between 16 and 45 nucleotides with at least 0.01 RPM in 100% of the PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 19 / 25 Human sperm displays rapid responses to diet samples. Data are available in S1 Data, SRA project: SRP065418, and SRA project: SRP132262. (EPS) S7 Fig. Mitochondrial tsRNAs show a diverse repertoire of cleavage sites. Graphs show the same coverage and cleavage-site analysis as was presented in Fig 5A–5D for nuclear tsRNA, but with the significant mitochondrial tRNA isodecoders reported in Fig 2H. Each isodecoder panel is divided into 3 subpanels: overall nucleotide coverage (upper panel), start-site expres- sion (middle panel), and end-site expression (lower panel). Each panel is summarized into mean RPM in Sugar (dark grey) and Healthy (light grey), respectively. Contrary to nuclear tRNAs, mitochondrial tRNAs do not show apparent coherency in cleavage-site location. Note, however, that they are still strongly co-regulated as shown by the cluster analysis in Fig 3 and scatterplot of Fig 6F. Data are found in S2 Data. (EPS) S8 Fig. Evidence for nitRNA cleavage in the TTCGA T-loop of a tRNA isodecoder with high content of 50 -tsRNAs. Graphs showing the difference between Sugar (red) and Healthy (blue) on nucleotide coverage across tsRNA types plotted over a GluCTC sequence. Upper panel show 50 -tsRNAs and 50 -halves, middle panel i-tsRNAs, and lower panel 30 -tsRNA and 30 - halves. The GluCTC isodecoder was not affected by a high-sugar diet when including all types of fragments mapping to it (Fig 2H). It was, however, identified to have significant nitRNAs in the T-loop cleavage analysis (S4 Table) (Fig 5J). Note that the 50 -tsRNAs and 50 -halves (upper panel) were unaffected by diet, explaining why it did not show any difference in the isodecoder analysis. In line with the T-loop analysis, however, many i-tsRNAs increased by high sugar (middle panel). Also note that the 30 -tsRNAs (lower panel), which are likely cleavage bypro- ducts when 50 -tsRNAs are generated, was unaffected by diet. Data are available in S2 Data. (EPS) S9 Fig. Diet-sensitive production of nitRNA. Most of the significantly changed nuclear tsRNAs had a 30 -end within the T-loop. The position of the 50 -start nucleotide was less precise, rather it varied with different types of tRNA isodecoder. The diet-sensitive cleavage would the- oretically give rise to a matching 30 -tsRNA. While there was some evidence for such matching 30 -tsRNAs (see Fig 5D, S4 Fig, S4 Table), they generally showed low expression. Possible expla- nations for the missing 30 -tsRNA involves selective degradation of 30 -byproducts, or that only nitRNAs (but not 30 -tsRNAs) are delivered to the sperm by exosome shuttling. An incompati- bility between RNA modifications present on the 30 -byproducts and the enzymes used in pre- paring the sequencing libraries is also a possible explanation. (EPS) S1 Table. Estimated physiological/metabolic parameters of the study participants (n = 15). Statistical summary of the BMRs, PALs, and TEEs of the participants at intervention start point. (XLSX) S2 Table. Phenotypic data and results. Contains raw data and statistical summaries for semen (including sperm motility), body and blood parameters of all participants. (XLSX) S3 Table. Changes in sperm miRNA following a high-sugar diet. Contains the DEseq2 results from a targeted analysis on miRNA annotated by Sports. (XLSX) S4 Table. Annotation and statistical summary for MINTmap analysis after filtration. Con- tains detailed annotations and statistical results from comparing Sugar versus Healthy diets on PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 20 / 25 Human sperm displays rapid responses to diet all analyzed tsRNA. Note that column “tsRNA type” is presented with the original MINTmap names, where tRF = tsRNA (e.g., 30 -tRF = 30 -tsRNA). (XLSX) S5 Table. More information about tsRNA in Fig 3. Contains the sequences, MINTmap IDs, and the genome coordinates for the tsRNA presented in Fig 3. Also contains the center scaled RPM differences between Sugar and Healthy diets for each participant, which were used to construct the cladogram in Fig 3. The numbers in the first column refer to the tsRNA number (1–54) in Fig 3. Note that the R script in S1 Text will regenerate the full analytical procedure for Fig 3. (XLSX) S1 Text. R script. Contains both the instructions and the actual code for automatically regen- erating all calculations and graphs presented in Figs 2 and 3 using the input data found in S1 and S2 Data. (R) S1 Data. Sports output data. Read counts generated by Sports. (XLSX) S2 Data. MINTmap output data. RPM of individual tsRNA extracted from MINTmap and normalized in relation to the total reads of the original raw sequence files (fastq). Note that col- umn “tsRNA type” is presented with the original MINTmap names, where tRF = tsRNA (e.g., 30 -tRF = 30 -tsRNA). (XLSX) S3 Data. qPCR data. RPM and Ct values generated in and prior to the qPCR validation exper- iment. (XLSX) S1 Raw Images. Original, uncropped, and minimally adjusted images. Contains the original photo of the gel electrophoresis used to verify the fragments lengths after the qPCR validation, as presented in S3D Fig. (TIF) Acknowledgments We would like to express our gratitude to the staff at restaurant Cellskapet, who made sure that every participant got personalized lunch/dinner boxes, to E. Öst for the illustration of the participants, and to A. Belmont for valuable feedback during manuscript preparation. Author Contributions Conceptualization: Daniel Nätt, Unn Kugelberg, Elizabeth Nedstrand, Stefan Zalavary, Marie Löf, Anita Öst. Data curation: Daniel Nätt, Eduard Casas, Anita Öst. Formal analysis: Daniel Nätt, Eduard Casas, Marie Löf, Anita Öst. Funding acquisition: Cristian Bellodi, Anita Öst. Investigation: Unn Kugelberg, Stefan Zalavary, Pontus Henriksson, Carola Nijm, Julia Jäder- quist, Johanna Sandborg, Eva Flinke, Rashmi Ramesh, Lovisa Örkenby, Filip Appelkvist, Thomas Lingg, Nicola Guzzi. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000559 December 26, 2019 21 / 25 Human sperm displays rapid responses to diet Methodology: Daniel Nätt, Unn Kugelberg, Eduard Casas, Thomas Lingg, Nicola Guzzi. Project administration: Unn Kugelberg, Anita Öst. Resources: Daniel Nätt, Cristian Bellodi, Anita Öst. Software: Daniel Nätt, Tanya Vavouri. Supervision: Unn Kugelberg, Elizabeth Nedstrand, Cristian Bellodi, Tanya Vavouri, Anita Öst. Validation: Daniel Nätt, Nicola Guzzi, Cristian Bellodi. Visualization: Daniel Nätt, Unn Kugelberg, Anita Öst. Writing – original draft: Daniel Nätt, Unn Kugelberg, Tanya Vavouri, Anita Öst. Writing – review & editing: Daniel Nätt, Unn Kugelberg, Eduard Casas, Elizabeth Nedstrand, Stefan Zalavary, Pontus Henriksson, Carola Nijm, Julia Jäderquist, Johanna Sandborg, Eva Flinke, Rashmi Ramesh, Lovisa Örkenby, Filip Appelkvist, Thomas Lingg, Marie Löf, Tanya Vavouri, Anita Öst. References 1. Nelson CMK, Bunge RG. 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