ИСТИНА

Изучить изменения эпигенетических маркеров, включая профиль фрагментов транспортной и рибосомальной РНК в сперматозоидах крыс и людей при воздействии возраста (крысы), характеристик пубератного периода и гормонального профиля (люди), а также при действии химических экспозиций хлорорганическими соединениями, свинцом и фталатами (люди) и ингибиторами горения (крысы).

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 aniandrogenic 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 ΣDiNP 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.

Мы ожидаем получить следующие результаты: 1. Возрастные изменения (на 65-й постнатальный день, пубертат и 120-й постнатальный день, половозрелость) профилей фрагментов тРНК (фтРНК) и фрагментов рРНК (фрРНК) в сперматозоидах крыс, их функциональное значение. 2. Изменения профилей фтРНК и фрРНК в сперматозоидах крыс в зависимости от перинатальной экспозиции бромированным антипиретиком БДЕ-47. 3. Сопряженные изменения метилирования ДНК с модификациями гистонов (по данным литературы) в ассоциированных участках генома крыс. 4. Влияние различных физиологических вариантов течения пубертата на эпигенетические маркеры сперматозоидов мужчин молодого возраста, включая метилирование ДНК и профиль малой РНК. 5. Влияние различных физиологических вариаций гормонального профиля (ЛГ, ФСГ, тестостерон, пролактин, ТТГ) на эпигенетические маркеры сперматозоидов мужчин молодого возраста, включая метилирование ДНК и профиль малой РНК.

Наши предыдущие исследования «Поиск эпигенетических маркеров воздействия химических веществ на геном мужских репродуктивных клеток и качество семени у человека и лабораторных крыс» связаны с изучением таких эпигенетических маркеров, как метилирование ДНК сперматозоидов (проект РНФ 14-45-00065) и профиля мнкРНК сперматозоидов (проект РНФ 18-15-00202) как физиологических (возраст), так и под действием различных химических веществ окружающей среды.