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Project titleEffects of endocrine disrupting chemiclas on epigenetic reprogramming of male human and rat germ cells and semen quality

AffiliationFederal State Budgetary Educational Institution of Higher Education Lomonosov Moscow State University,

Research area 05 - FUNDAMENTAL RESEARCH IN MEDICINE, 05-401 - Molecular and cellular medicine

Keywordssmall noncoding RNA, DNA methylation, semen quality, epigenetics, reproductive health, dioxins, lead, longitudinal study, cohort, sperm epigenome, molecular epidemiology, epigenetic epidemiology, endocrine disrupting chemicals, spermatogenesis

Annotation
In our previous research we analyzed epigenetic markers in spermatozoa, including DNA methylation (RSF14-45-00065) and sncRNA (RSF 18-15-00202), in relation to physiological parameters (age) and environmental exposures. The relevance of these studies is determined by the high incidence of paternal infertility (Inhorn & Patrizio, 2015), negative trends for sperm quality over the last 35 years (Levine et al., 2017), accumulating evidence on negative effects of environmental exposures on sperm quality, and, specifically, on sperm epigenome (Marcho et al., 2020) – changes that can be transferred to the next generation (Natt & Ost, 2020; Wei et al., 2015). The goal of this proposed extension of our research is to analyze changes in epigenetic markers, including profiles of fragments of tRNA (ftRNA) and rRNA (frRNA) in spermatozoa of humans and rats in response to natural aging (rats), trajectories of pubertal development and variation of hormone profile (humans), and chemical exposures to chlororganic compounds, phthalates and led (humans) and brominated flame retardants (rats). In the current application, we add new factors that can affect sperm epigenome as well as new epigenetic markers (Tables 1 and 2). Among chemical exposures, we will focus on phthalates – ubiquitous environmental contaminants. Data form our cohort study “Russian Children’s Study” for the first time demonstrate that children and adolescents in Russia are exposed to higher phthalate doses than same age groups in the USA and Germany. These exposures include phthalates with ani-androgenic properties (Table 1). According to a systematic review, DEHP and DBP affect sperm quality and testosterone levels in adults (Radke et al., 2018). Our data show that higher urinary concentrations of ∑DiNP in the late pubertal samples were associated with poorer semen quality (Mínguez-Alarcón et al, 2020). Members of our research group (Suvorov, Pilsner) also showed that among patients of infertility clinic, phthalates are associated with altered spermatozoa methylome and poor embryo quality (Wu, Ashcraft, et al., 2017; Wu, Estill, et al., 2017). In spite of rapidly increasing number of sperm epigenome studies it is unclear, if observed changes are normal physiological, compensatory, stochastic or programed by natural selection. Our data on sperm epigenome changes, including DNA methylation (Pilsner et al., 2020), and sncRNA (Suvorov et al., 2020) demonstrated that effects of chemical exposures can be dissected only when normal physiological changes (age related for example) are accounted for. For example, nothing is known about the role of physiological parameters of pubertal period (puberty initiation, progress and sexual maturity onset) for sperm epigenome. Nothing is known as well about the role of major reproductive hormones, LH, FSH, testosterone on sperm epigenetics in the population of healthy men. Based on the data that have been accumulated in a framework of the parent «Russian Children’s Study”, we plan to study for the first time effects of • exposure to phthalates during 4 sensitive windows of peripubertal development (pre-pubertal, early puberty, mid to late puberty and sexual maturity), • physiology of pubertal development; • physiological variation in hormonal profiles; on epigenetic changes in spermatozoa, which data were produced in two previous RSF projects. Another aspect of our study added as new at that step, consists in a new approach for the analysis and interpretation of data on frRNA and ftRNA. During our work on the previous project, we mostly focused on the analysis miRNA and piRNA – small RNAs with well-recognized epigenetic and functional roles. We also detected ftRNA and frRNA in both rat and human studies. In human spermatozoa ftRNA and frRNA fractions accounted for 68% of all mapped sncRNA (Fig. 1), what is concordant with other studies, showing prevalence of ftRNA and frRNA fractions in mature spermatozoa (Donkin et al., 2016; Hua et al., 2019; Natt & Ost, 2020). Additionally we demonstrated significant, age-dependent decrease in frRNA fraction in rats from 46% in young rat to 37% in mature; and significant, age-dependent increase in ftRNA fraction from 10% in young rat to 17% in mature animals (Fig. 2) (Suvorov et al., 2020). In both species, humans and rats, we also registered ftRNA and frRNA associated with chemical exposures. Further interpretation of these data were hindered by the luck of knowledge about functions in a cell of these RNA subtypes, their role in epigenetic information transfer via germ-line as well as shortage in available methodological approaches for ftRNA and frRNA analysis. Understanding that ftRNA and frRNA are not some random products of tRNA and rRNA degradation, but rather represent new classes of regulatory molecules, with important cellular functions, emerged only recently. Recent studies demonstrated that mature tRNA and pre-tRNA undergo fragmentation into several specific types of products: small RNA, fragments of tRNA and halves of tRNA (Shen et al., 2018; Zhu et al., 2019). FtRNA and frRNA may participate in epigenetic information transfer via spermatozoa. For example, in one study male mice fed with high-fat diet produced spermatozoa with altered composition of ftRNA (Chen et al., 2016). Offspring of these fathers developed decreased glucose tolerance and insulin sensitivity. Injection of ftRNA altered by high-fat diet into fertilized egg of control animals resulted in similar changes in embryo metabolism. Recent studies show, that micro-vesicles of epididymis (epididymosomes) deliver a cargo of ftRNA and frRNA to mature spermatozoa (Trigg et al., 2019), and the content of this cargo may affect sperm competition, fertilization, embryo development and transgenerational inheritance (Conine et al., 2018; Sharma et al., 2016; Sharma et al., 2018). Approaches for ftRNA and frRNA quantification and functional analysis were developed only recently (Natt et al., 2019; Xie et al., 2020). In the current project, we will focus on the analysis of ftRNA and frRNA profiles and changes of these profiles in response to several factors. Only 1-3 other laboratories in the world have expertise, experience and data to accomplish similar goals. Thus, our project will capitalize on the data on effects of chemical exposures on molecular-epigenetic mechanisms in spermatozoa from our previous RSF projects (DNA methylation - RSF14-45-00065, sncRNA - RSF 18-15-00202), as well as the data on pubertal trajectories, hormonal profiles and exposures to phthalates during 4 sensitive peripubertal windows to dissect how spermatozoa epigenome, including profiles of ftRNA and frRNA, is shaped by the interaction of natural, physiological and environmental factors.

Expected results
The following results will be produced: 1. Age-dependent changes in ftRNA and frRNA in rat sperm and functional significance of these changes. 2. Changes of ftRNA and frRNA profiles in rat sperm in response to perinatal exposure to BDE-47. 3. Concordant changes in DNA-methylation and histone modification (literature data) in rat sperm. 4. Associations between pubertal trajectories and spermatozoa epigenome in young men, including profiles of DNA methylation and sncRNA. 5. Associations of hormonal levels (LH, FSH, testosterone) and spermatozoa epigenome in young men, including profiles of DNA methylation and sncRNA. 6. Associations between phthalate exposures during 4 critical peripubertal periods (prepubertal, early puberty, middle stages of puberty, sexual maturity) and spermatozoa epigenome in young men, including profiles of DNA methylation and sncRNA. 7. Associations between EDCs, including chlororganic compounds (dioxins, furans, PCBs, insecticides), led, phthalates and profiles of ftRNA and frRNA in spermatozoa of young men. 8. Associations between semen quality and ftRNA and frRNA profiles in young men. Analysis of epigenetic profiles of spermatozoa in relation to pubertal trajectories is a fundamental goal in the understanding of developmental physiology of male reproductive system. Interaction between circulating hormones and endocrine disruptors and their role in shaping sperm epigenome is also an important and complex goal. Exposures to phthalates have global character, and majority of the general population is exposed to these compounds. Therefore, effects of phthalates on sperm epigenome - information that can be transferred to the next generation - is an important goal for the regulatory decision-making related to phthalate production and use. Additionally to relatively well-studied epigenetic changes in spermatozoa (DNA methylation, miRNA), in our project, we plan to focus on ftRNA and frRNA which are an emerging new classes of regulatory molecules with specific epigenetic functions in spermatozoa. The discovery of new classes of regulatory molecules is always associated with an explosive progress in different areas of medico-biological science. Such discoveries build opportunities for new diagnostics approaches, establishing of new links between environmental factors and biological processes, uses of new molecules as therapies and as targets of therapeutic interventions, use of new molecules as biomarkers of different effects, states and conditions of the organisms, and finally opens doors for better understanding of fundamental principles of living systems. In particular, our project will identify factors affecting ftRNA and frRNA in spermatozoa and will establish their link with reproductive health. That will open an opportunity to use these molecules as diagnostic biomarkers of male fertility. In long-term our study will allow identifying ftRNA and frRNA - therapeutic targets for the prevention of male fertility and abnormalities in embryo development. Using our animal model, we will analyze epigenetic aging of sperm. That line of research will build a background for the development of interventions preventing epigenetic aging. Our study provides a unique opportunity to move forward our understanding of interaction between methodologically accessible changes in sperm epigenome (DNA methylation, sncRNA, including ftRNA and frRNA) with different environmental factors and men reproductive health. First, it is based on 2 organisms (humans and rats), allowing for the use of different complementary methodological approaches (analysis of associations in human populations and laboratory experiment with rats) to establish common phenomena. Second, the human cohort is very well described and includes detailed data on chemical exposures, socially-demographic and life-style parameters, detailed analysis of reproductive development, characteristics of reproductive health, and epigenetic profiles of spermatozoa. Third, our rat model also includes chemical exposure, age factor, detailed information on reproductive health and epigenetic profiles in spermatozoa.

Annotation of the results obtained in 2022
The goal of this study in the current year was to analyze epigenetic markers (profiles of small non-coding RNA (sncRNA) including fragments of tRNA (ftRNA)) of ejaculate quality as well as markers of chemical exposures in the sperm of 18-year-old men. The following chemical exposures were included in our study: organochlorine compounds and lead during the peripubertal period, and phthalates at 4 sensitive windows of pubertal development: prepuberty, early puberty, late puberty, and maturity. Optimization of bioinformatic pipelines. Using the benchmarking approach, we reviewed existing methods for the analysis of sncRNA profiles including ftRNAs. This analysis covered parameters for read trimming, filtering, mapping, transcript quantification, and differential expression; and was done using datasets from 7 published studies including 4 focusing on spermatozoa (Donkin et al., 2016, Ingerslev et al., 2018, Hua et al., 2019, Morgan et al., 2020). Based on the benchmarking analysis we build an optimized bioinformatic pipeline. This pipeline includes the following settings (1) low bound for reads’ trimming at 15 nt and the upper bound set at read length minus 40% of adapter length; (2) use of bowtie with one mismatch allowed; (3) use of the Integrated Transcript Annotation for Small RNA (ITAS) database, a database developed by our group (Stupnikov et al., 2022); 4) filtering by a median read number per transcript > 5; (5) use of DESeq2 for differential expression analysis. The following pipeline was optimized for ftRNA analysis: (1) filtered reads mapping to the reference genome; (2) selection of reads mapped to tRNA; (3) expression analysis using kallisto with k-mer size = 11. This line of research focusing on bioinformatic pipelines optimization resulted in a manuscript available via Preprint and submitted to a Q1 (Scopus) journal: «Approaches for sRNA analysis of RNA-seq data: comparison, benchmarking» (Bezuglov et al., 2022). In our protocols, we used gradient centrifugation to separate fractions of spermatozoa with different densities, where higher density corresponds to a higher maturity (Malvezzi et al., 2014). It was demonstrated previously, that sncRNA profiles are different in these fractions (Capra et al., 2017) as mature spermatozoa undergo enrichment with sncRNA via extracellular vesicles in epididymis (Sharma et al., 2018). Therefore, we stratified our analysis of sncRNA by density fractions. In the fraction of mature spermatozoa, we identified 344 sncRNAs with read counts > 5: 116 mature miRNAs, 191 piRNAs, and 37 ftRNAs. In the fraction of less dense spermatozoa, we identified 393 sncRNAs with read counts > 5: 99 mature miRNAs, 247 piRNAs, and 47 ftRNAs. Phthalate exposures during 4 sensitive windows of pubertal development and changes in the sperm epigenome. We analyzed associations between 15 individual phthalate metabolites as well as their groups based on chemical structures and biological activities [ΣDEHP = (MEHP + MEHHP + MEOHP + MECPP), ΣDiNP = (MHiNP + MOiNP + MCOP), ΣDiDP = (MHiDP + MOiDP + MCNP), Σ Anti-androgenic phthalates (AAP) = (MEHP + MEHHP + MEOHP + MECPP + MnBP + MiBP + MBzP + MHiNP + MOiNP + MCOP)] with epigenetic changes in sperm, including profiles of sncRNAs, and DNA-methylation. Effects of exposure at 4 different windows (prepuberty, early puberty, late puberty, and maturity) were analyzed. Exposure during each period resulted in significant changes in methylation of many specific CpGs and in expression changes of some sncRNAs in 18-year-old men. For example, exposure during prepuberty resulted in expression changes of 52 and 34 sncRNA in mature and less mature spermatozoa respectively with FDR-adjusted p < 0.05. Similarly, exposure during early puberty, late puberty, and maturity affected 78 and 19, 24 and 26, and 26 and 26 sncRNA respectively. Exposures during prepuberty and early puberty have a stronger effect on sncRNA in mature spermatozoa, while exposures during late puberty and maturity have a stronger effect on sncRNA in immature sperm. Among individual phthalates, the strongest effects were seen for monoethyl phthalate (MEP), where 39 and 10 differentially expressed sncRNA were identified in mature spermatozoa following exposure at pre- and early puberty respectively. Similarly, 47 differentially expressed sncRNA were identified in immature spermatozoa following exposure to MEP at late puberty. Forty-five differentially expressed sncRNA changed their expression in response to at least 4 phthalate metabolites. “Cell adhesion molecules” was identified as a top enriched pathway (-log10(P)=13) in functional analysis with genes-targets of the top differentially expressed sncRNA. tRF-19-Q9B1XSI9_21107 was one individual ftRNA negatively associated with ΣDiNP in urine during late puberty. This molecule is an i-tRF fragment of CysGCA tRNA 19 nt in length (GCTTCAAACCTGCCGGGGC). We demonstrated earlier that phthalate exposures during this period are negatively associated with sperm quality (Minguez-Alarcon et al., 2022). Interestingly expression of this fragment in mature spermatozoa was positively associated with blood LH, testosterone, and TSH. In immature spermatozoa, it was also positively associated with sperm quality parameters and testes size. Functional analysis of 579 target genes of this RNK using piRanha (Jehn et al., 2020), retrieved androgenic signaling as the top enriched category (-log10(P)=3.7). No information was found in the published literature to describe the biological role of this RNA. Thus, our analysis for the first time identifies ftRNA - i-tRF CysGCA as a marker of functional events in spermatozoa, including their response to chemical exposures during the peripubertal period and physiological processes controlled by LH and testosterone. The multifactorial analysis allowed us to generate a matrix of DNA methylation response to phthalate exposures during 4 windows of pubertal development. Exposures during pre-, early, and late pubertal periods and maturity resulted in 2797, 2385, 3434, and 1275 differentially methylated CpGs (p < 0.005) respectively. Twenty-five CpGs were differentially methylated in response to at least 4 phthalate metabolites at one period of pubertal development. For example, CpG chr21_33019721 responds to late pubertal exposure to ΣDEHP metabolites, ΣDiNP metabolites, and androgenic phthalates. We demonstrated previously that ΣDiNP exposure during late puberty is negatively associated with sperm quality (Minguez-Alarcon et al., 2022). Our current analysis shows that exposure to ΣDiNP during late puberty results in the highest number of differentially methylated CpGs (478) as compared with 152, 123, and 73 CpG following prepubertal, early pubertal, exposures and exposure during maturity respectively. These results support specific sensitivity of the late puberty (testes size ≥10 - 15 мл, age = 13.13 years) for phthalate exposures. Organochlorine compounds, lead, and sncRNA profiles in spermatozoa. Our analysis identified changes in the expression of sncRNA following peripubertal (8-9 years of age) exposure to dioxins, furans, dioxin-like PCBs, non-dioxin-like PCBs, and organochlorine pesticides. Immature spermatozoa demonstrated higher numbers differentially expressed sncRNA in response to exposures. For example, the numbers of these RNAs were as follows for different compounds and mixtures: 16 for the sum of dioxin-like compounds, 9 for dioxin equivalent, 9 for the sum of mono-ortho PCBs, 15 for DDT metabolite ppDDE, and 8 for hexachlorocyclohexane (FDR adjusted p < 0.05). Five sncRNAs were differentially expressed following peripubertal exposure to lead in immature spermatozoa. We identified 13 sncRNAs (including 3 ftRNAs: tRF-46-87R8WP9N1EWJQ72S3H0_99, tRF-38-FYRE987S1RJ7RMDV_834, and tRF-27-59KNNVORRNK_6930), which were significantly associated with at least 5 organochlorine compounds (FDR adjusted p < 0.1). Hsa-piR-22273 was among the most often responders to organochlorine exposures (12 compounds, FDR adjusted p < 0.1). It was also significantly associated with peripubertal exposure to lead (FDR adjusted p = 0.02). Hsa-piR-15181 was another piRNA associated with both lead and organochlorine exposures. It responded to phthalate exposures as well. Both piRNAs are functionally enriched for “pregnancy”. sncRNA and ejaculate quality. We identified sncRNA sassociated with sperm quality. Interestingly, higher numbers of such sncRNAs were found in immature spermatozoa: 13 associated with sperm concentration and 10 with the number of progressively motile spermatozoa (FDR adjusted p < 0.05). We also identified 7 sncRNAs, including five ftRNAs (tRF-43-17NSRJ7KYUHR0VX6D2_311, tRF-19-FZ2DN1IY_21688, tRF-19-Q9B1XSI9_21107, tRF-29-79MP9P9NH52Q_4926, and tRF-45-7Z2R1HPSR9O9337KB6_151), significantly associated with at least 5 ejaculate parameters (FDR adjusted p < 0.10). miRNAs hsa-miR-320c and hsa-miR-320b are positively associated with the number of progressively motile spermatozoa in immature spermatozoa as well as other sperm parameters. Functional analysis of genes-targets of hsa-miR-320c identified “cell adhesion molecules” as a highly enriched biological category (-log10(P)=17). The same category is enriched by genes-targets of transcripts differentially expressed in response to phthalate exposures. It was shown previously that increased expression of miR-320 in Sertoli cells results in decreased sperm quantity (Zhang et al., 2018). sncRNA variability in paired ejaculates. Variability was evaluated for 188 miRNAs, 930 piRNAs, 326 rRNAs, and 128 tRNAs in 10 paired ejaculates collected at one-week intervals using calculation of the average difference between pairs, Wilcoxon pairwise comparison, variation coefficients, contingency correlations, and Bland-Altman analysis. We observed moderate contingency of sncRNA in paired ejaculates with rho = 0.71 for tRNA, 0.65 for piRNA, and 0.54 for miRNA. We also identified some highly variable sncRNAs including 3 piRNAs and 1 miRNA, p < 0.005. As a result of this project, we accomplished an analysis of the effects of chemical exposures on semen quality and published papers in Q1 (WOS) journals, including «Association of peripubertal blood lead levels with reproductive hormones and semen parameters in a longitudinal cohort of Russian men» (Williams et al., 2022) and «Urinary phthalate metabolite concentrations during four windows spanning puberty (prepuberty through sexual maturity) and association with semen quality among young Russian men» (Minguez-Alarcon et al., 2022). We also have published 2 review papers in Q1 (WOS) journals, including «Mechanisms of Male Reproductive Toxicity of Polybrominated Diphenyl Ethers» (Arowolo et al., 2022), and «Age-associated epigenetic changes in mammalian sperm: implications for offspring health and development» (Ashapkin et al., 2022). In the current year our multidisciplinary team presented 4 presentations, including 3 oral talks and one poster at the 12th European Congress of Andrology, October 19-21, 2022, Barcelona, Spain (https://eca2022.com/); at the 13th International Multiconference “Bioinformatics of Genome Regulation and Structure/Systems Biology”, July 4-8, 2022, Novosibirsk (https://bgrssb.icgbio.ru/2022/); at the International Conference КАРМ-13 "Key Aspects of Reproductive Medicine", Moscow, June 2-5, 2022; and at the XXXII Annual International Conference РАРЧ "Reproductive Technology Today and Tomorrow", Kazan, September 7-10, 2022.

Publications

1. Bezuglov V., Stupnikov A., Skakov I., Shtratnikova V. , Pilsner J.R., Suvorov A., Sergeyev O. Approaches for sRNA analysis of RNA-seq data: comparison, benchmarking Preprints, 2022120177 (year - 2022) https://doi.org/10.20944/preprints202212.0177.v1

2. Bezuglov V., Stupnikov A., Skakov I., Shtratnikova V. , Pilsner J.R., Suvorov A., Sergeyev O. Approaches for sRNA analysis of RNA-seq data: comparison, benchmarking International Journal of Molecular Sciences, 24(4):4195 (year - 2023) https://doi.org/10.3390/ijms24044195

3. Minguez-Alarcon L., Burns J., Williams P. L., Korrick S. A., Lee M. M., Bather J. R., Kovalev S. V., Sokolov S. A., Lebedev A. T., Smigulina L., Ghayda R. A., Koch H. M., Sergeyev O., Hauser R. Urinary phthalate metabolite concentrations during four windows spanning puberty (prepuberty through sexual maturity) and association with semen quality among young Russian men International Journal Of Hygiene And Environmental Health, 243, 113977 (year - 2022) https://doi.org/10.1016/j.ijheh.2022.113977

4. Stupnikov A., Bezuglov V., Skakov I., Shtratnikova V., Pilsner J. R., Suvorov A., & Sergeyev O. ITAS: Integrated Transcript Annotation for Small RNA Non-coding RNA, 8(3):30 (year - 2022) https://doi.org/10.3390/ncrna8030030

5. Arowolo O., Pilsner J. R., Sergeyev O., & Suvorov A. Mechanisms of Male Reproductive Toxicity of Polybrominated Diphenyl Ethers International Journal of Molecular Sciences, 23, 14229 (year - 2022) https://doi.org/10.3390/ijms232214229

6. Ashapkin V., Suvorov A., Pilsner J. R., Krawetz S. A., Sergeyev O. Age-associated epigenetic changes in mammalian sperm: implications for offspring health and development Human Reproduction Update, dmac033 (year - 2022) https://doi.org/10.1093/humupd/dmac033

7. Bezuglov V., Stupnikov A., Skakov I., Shtratnikova V. , Pilsner J.R., Suvorov A., Sergeyev O. ITAS: integrated transcript annotation for small RNA Abstracts the Thirteenth International Multiconference,BGRS/SB-2022, p.68-69 (year - 2022) https://doi.org/10.18699/SBB-2022-031

8. Sergeyev O., Ashapkin V., Suvorov A., Pilsner J. R., Krawetz S. A. Age-associated epigenetic changes in mammalian sperm: implications for offspring health and development ANDROLOGY. 12-th European Congress of Andrology Abstract Book, 10, 3, 68-69 (year - 2022)

9. Suvorov, Alexander; Pilsner, J. Richard; Dribnokhodova, Olga; Dunaeva, Elena; Gerasimov, Evgeny; Logacheva, Maria; Marcho, Chelsea;Naumov, Vladimir; Shershebnev, Alex; Shtratnikova,Victoria; Wu,Haotian; Zheludkevich, Anna; Sergeyev, Oleg Aging Induces Profound Changes in Rat Sperm Epigenome and These Changes are Modified by Perinatal Exposure to Environmental Flame Retardant 21-st European Testis Workshop. Miniposters book, 88 (year - 2021)

10. Victoria Shtratnikova, Vladimir Naumov, Vitaly Bezuglov, Luidmila Smigulina, Tatiana Denisova, Yury Dikov, Alexander Suvorov, J. Richard Pilsner, Russ Hauser, Stephen A.Krawetz, Oleg Sergeyev Peripubertal serum concentrations of dioxins and sperm small non-coding rnas in the prospective cohort of healthy young men 21-st European Testis Workshop. Miniposters book, 67 (year - 2021)

11. - ПОЗДНЕЕ ОТЦОВСТВО ИЗМЕНЯЕТ ЭПИГЕНОМ СПЕРМАТОЗОИДОВ И МОЖЕТ ВЛИЯТЬ НА ЗДОРОВЬЕ ПОТОМКОВ Научная Россия, 14.10.2022 12:30 (year - )

12. - Эпигенетические изменения спермы возрастных мужчин могут влиять на здоровье их детей Индикатор.ру, 22:19, 14 ОКТЯБРЯ 2022 (year - )

13. - Российские ученые выяснили, как определить возраст сперматозоидов Газета.ру, 14 октября 2022, 12:10 (year - )

Annotation of the results obtained in 2021
The goal of this study is to analyze changes in epigenetic markers, including profiles of fragments of tRNA and rRNA, in spermatozoa of rats and humans in relation to age (rats), characteristics of pubertal period and hormonal profiles (humans), exposures to organochlorine compounds, led, and phthalates (humans), and flame retardants (rats). During the first year, we focused on the development and optimization of bioinformatic approaches for the analysis of small non-coding RNA (sncRNA), including fragments of tRNA (ftRNA) and fragments of rRNA (frRNA), detected using next generation sequencing (NGS). Existing methods use sequential mapping of reads on transcripts from different databases (Shi et al., 2018). This approach was used in research focusing on spermatozoa as well (Nätt et al., 2019; Zhang et al., 2021). This approach has several disadvantages. To overcome these disadvantages we integrated existing databases, made quality analysis of existing data, and fixed many existing problems. In particular, all loci were converted into coordinates of one version of genome, transcripts’ sequences were verified to match corresponding loci, transcripts with overlapping loci were filtered out, annotations were all formatted in accordance with gtf format, multi-loci transcripts were identified as multi-feature. Several pipelines were tested for the analysis of expression, using Rsubread, RSEM and Kallisto, using our and publicly available literature data (Donkin et al., 2016; Ingerslev et al., 2018). Based on the ability of pipelines to produce concordant results between each other and with literature data and their ability to detect different types of RNA, two pipelines were selected for further use: one based on Rsubread and another based on Kallisto with the length of k-mer 11. For ftRNA and frRNA analysis an existing instrument SPORTS was adapted to fix a problem of transcript annotations. Differential expression of sncRNA and fragments was analyzed using DESeq2 (Love et al., 2014). This choice is motivated by the ability of DESeq2 to analyze data with complicated structure, the need to compare groups with covariate variables, and its stability (Stupnikov et al., 2021). For human data MINTBase vas used as a source of data for ftRNA. Analysis of ftRNA and frRNA in rat sperm did not identify any changes associated with chemical exposure. However, changes associated with age were profound and we have found that profiles of ftRNA and frRNA are good predictors of age. Different bioinformatic approaches identified highly abundant and age-dependent ftRNAs and frRNAs that mapped to 36 mature tRNAs and one mature rRNA (union). Both pipelines identified as differentially expressed 8 mature tRNAs and one mature rRNA (intersection). Interestingly, two age dependent tRNAs in rat spermatozoa (tRNA-Gln-CTG and tRNA-Gln-TTG) were also differentially expressed in humans in our study in relation to different pubertal trajectories and FSH levels in blood. Fragments of one of these tRNAs are also age-dependent in mice (Guo et al., 2021). We analyzed intersection between different age-dependent epigenetic mechanisms in rat spermatozoa using our and literature data. Coordinates of age-dependent differentially methylated regions of DNA (DMRs) intersected significantly with loci of coordinates of age-dependent sncRNAs, suggesting that some age-dependent changes in different epigenetic mechanisms may be causally linked. We also analyzed intersection between age-dependent DMRs with DNA regions associated with retained histones (DRH). (Most of histones are substituted by protamines in spermatozoa to insure high compactization of chromatin.) This analysis identified 1330 genes associated with both, age-dependent DMRs and DRH. Major biological categories enriched with these genes were relevant to embryonic development. Additionally, enriched disease categories included these pathologies in children, which were shown to be associated with advanced paternal age. These categories include cleft palate, childhood leukemia, blastomas, and neurodevelopmental and psychiatric disorders. Thus, nucleosomes mark age-dependent DMRs which play important role in the regulation of development. These regions may be used as potential therapeutic targets to address health of offspring conceived by parents of advanced age. We reanalyzed sncRNA from spermatozoa of 49 man-subjects using our optimized library preparation (published in (Shtratnikova et al., 2021)) and bioinformatic approaches. sncRNAs were filter using Rsubread and Kallisto with k-mer=11 to decrease false-positive findings keeping high level of sncRNA detection. Based on optimization experiments Rsubread pipeline was selected with a threshold of detection > 5 reads in at least half of all samples. The following numbers of sncRNA (330 total) were selected for the differential analysis using this approach: 101 miRNA, 167 piRNA, 4 rRNA, 35 mature tRNA, and 23 ftRNA. We obtained data on DNA methylation in a framework of our previous funded project (РНФ 14-45-00065). This analysis based on 36 subjects identified 47 531 CpG in all samples. Trajectories of pubertal development were determined for the whole cohort (n=489) of the Russian Childen’s Study based on yearly orchidometry starting 8-9 till 18-19 years of age (Plaku-Alakbarova et al, in press). Forty-nine subjects included in the current study were selected based on pubertal trajectories: slow (n=22, 45%), moderate (n=18, 37%) and faster (n=9, 18%). Teenagers/young men from “slow” group had slow start of testicles increase, smaller testicle size at 18-19 and poorer sperm quality (lower concentration and motility of spermatozoa) as compared with two other groups (Sergeyev et al, in preparation). Circulating FSH at 18-19, collected during the same visit as sperm was collected, correlated negatively with pubertal trajectories (rho = 0.55). We analyzed effect of pubertal trajectories on DNA methylation and sncRNA, including ftRNA and frRNA in spermatozoa of participants. Levels of several hormones (FSH, TSH, estradiol, free testosterone/estradiol ratio, testosterone/LH ratio) were associated with changes in expression of miRNA, piRNA, ftRNA, and DNA methylation in spermatozoa. We identified ftRNAs differentially expressed in relation to pubertal trajectories and FSH levels in circulation. These ftRNAs map on two mature tRNAs: tRNA-Gln-CTG и tRNA-Gln-TTG. Remarkably, same ftRNAs were also found to be significantly age-dependent in rat spermatozoa in the current study. Additionally, according to literature data, fragments of tRNA-Gln-TTG are linked to sperm quality in men (Chen et al., 2021), and play important role in the embryo development (Chen et al., 2020). In the current year, our multidisciplinary international team presented two oral talks at the 21st European Testis Workshop (https://etw2021.org/): “Peripubertal serum concentrations of dioxins and sperm small non-coding RNAs in the prospective cohort of healthy young men“ and “Aging Induces Profound Changes in Rat Sperm Epigenome and These Changes are Modified by Perinatal Exposure to Environmental Flame Retardant”. Another talk was presented at the QIAGEN Day in Moscow (https://www.youtube.com/watch?v=onwNqLnbHhM): “Spermatozoa epigenome: studies of age-dependent changes”. A visit of Drs. Alexander Suvorov and J.Richard Pilsner of the University of Masachusetts in Amherst, USA, members of the project research team, was organized in October 2021 to participate in 4 seminars. A manuscript review paper entitled “Age-associated epigenetic changes in mammalian sperm: implications for offspring health and development” was submitted to the Human Reproduction Update (Q1).

Publications

1. Shtratnikova V., Naumov V., Bezuglov V., Zheludkevich A., Smigulina L., Dikov Y., Denisova T., Suvorov A., Pilsner J.R., Hauser R., Krawetz S.A., Sergeyev O. Optimization of small RNA extraction and comparative study of NGS library preparation from low count sperm samples Systems Biology in Reproductive Medicine, Том 67, Выпуск 3, Страницы 230 - 243 (year - 2021) https://doi.org/10.1080/19396368.2021.1912851

2. Williams P. L., Minguez-Alarcon L., Korrick S. A., Lee M. M., Plaku-Alakbarova B., Burns J. S., Smigulina L., Dikov Y., Abou Ghayda, R., Hauser R., Sergeyev O. Association of peripubertal blood lead levels with reproductive hormones and semen parameters in a longitudinal cohort of Russian men Human Reproduction, - (year - 2022)

3. - Наследственная программа спермы существенно меняется с возрастом и под воздействием загрязнений окружающей среды NakedScience, - (year - )