Eduardo Larriba

Role of Non-Coding RNAs in the Transgenerational Epigenetic Transmission of the Effects of Reprotoxicants

Non-coding RNAs (ncRNAs) are regulatory elements of gene expression and chromatin structure. Both long and small ncRNAs can also act as inductors and targets of epigenetic programs. Epigenetic patterns can be transmitted from one cell to the daughter cell, but, importantly, also through generations. Diversity of ncRNAs is emerging with new and surprising roles. Functional interactions among ncRNAs and between specific ncRNAs and structural elements of the chromatin are drawing a complex landscape. In this scenario, epigenetic changes induced by environmental stressors, including reprotoxicants, can explain some transgenerationally-transmitted phenotypes in non-Mendelian ways. In this review, we analyze mechanisms of action of reprotoxicants upon different types of ncRNAs and epigenetic modifications causing transgenerationally transmitted characters through germ cells but affecting germ cells and reproductive systems. A functional model of epigenetic mechanisms of transgenerational transmission ncRNAs-mediated is also proposed.

Chronic low-dose exposure to a mixture of environmental endocrine disruptors induces microRNAs/isomiRs deregulation in mouse concomitant with intratesticular estradiol reduction

Humans are environmentally exposed not only to single endocrine-disrupting chemicals (EDCs) but to mixtures that affect their reproductive health. In reproductive tissues, microRNAs (miRNAs) are emerging as key targets of EDCs. Here, we analysed changes in the testis “miRNome” (and their biogenesis mechanism) in chronically exposed adult mice to a cocktail of five EDCs containing 0.3 mg/kg-body weight (BW)/day of each phthalate (DEHP, DBP, BBP) and 0.05 mg/kg-BW/day of each alkylphenol (NP, OP), from conception to adulthood. The testis “miRNome” was characterised using next-generation sequencing (NGS). Expression levels of genes involved in miRNA biogenesis were measured by RT-qPCR, as well as several physiological and cytological parameters. We found two up-regulated, and eight down-regulated miRNAs and thirty-six differentially expressed isomiRs along with an over-expression of Drosha, Adar and Zcchc11. A significant decrease of intratesticular estradiol but not testosterone was detected. Functional analysis showed altered spermatogenesis, germ cell apoptosis and negative correlation of miR-18a-5p with Nr1h2 involved in the deregulation of the steroidogenesis pathway. Here, we present the first association between miRNA/isomiRs deregulation, their mechanisms of biogenesis and histopathological and hormonal alterations in testes of adult mice exposed to a mixture of low-dose EDCs, which can play a role in male infertility.

Editor’s Highlight: Differential Effects of Exposure to Single Versus a Mixture of Endocrine-Disrupting Chemicals on Steroidogenesis Pathway in Mouse Testes

Endocrine-disrupting chemicals (EDCs) generate reproductive dysfunctions affecting the biosynthesis of steroid hormones and genes of the steroidogenic pathway. EDCs effects are mainly reported as a result of exposure to single compounds. However, humans are environmentally exposed to a mixture of EDCs. Herein, we assess chronic exposure to single alkylphenols and phthalates versus a mixture in mouse testes histology and steroidogenesis. Pregnant mice were exposed through drinking water to: 0.3 mg/kg-body weight (BW)/d of each phthalate (bis (2-ethylhexyl) phthalate, dibutyl phthalate, benzyl butyl phthalate), 0.05 mg/kg-BW/d of each alkylphenol (4-nonylphenol, 4-tert-octylphenol), or their mixture, covering from 0.5 postcoital day to weaning, continuing in the male offspring each exposure until adulthood (60-days old). Body and relative testis weight were increased in mixture-exposed mice along with histological alterations. Intratesticular testosterone (T) changed only in mice exposed to DBP, whereas estradiol (E2) levels were altered in all groups (except benzyl butyl phthalate). mRNA levels of genes encoding hormones of the steroid pathway (Cyp11a1, Hsd3b1, Cyp17a1, and Cyp19a1), cholesterol transporters (Star), and transcriptional factors (Sp1) showed that mice exposed to single or mixed compounds had alterations in at least 2 transcripts. However, none of the different types of exposure induced changes in all transcripts. In addition, changes at the mRNA or protein levels with single compounds were not always the same as those with a mixture. In conclusion, the effects of a chronic exposure to a mixture of EDCs on the expression of genes and proteins of the steroidogenic pathway and hormonal status were different from those exposed to single EDC.

Differential effects of exposure to single versus a mixture of endocrine-disrupting chemicals on steroidogenesis pathway in mouse testes

Endocrine-disrupting chemicals (EDCs) generate reproductive dysfunctions affecting the biosynthesis of steroid hormones and genes of the steroidogenic pathway. EDCs effects are mainly reported as a result of exposure to single compounds. However, humans are environmentally exposed to a mixture of EDCs. Herein, we assess chronic exposure to single alkylphenols and phthalates versus a mixture in mouse testes histology and steroidogenesis. Pregnant mice were exposed through drinking water to: 0.3 mg/kg-body weight (BW)/d of each phthalate (bis (2-ethylhexyl) phthalate, dibutyl phthalate, benzyl butyl phthalate), 0.05 mg/kg-BW/d of each alkylphenol (4-nonylphenol, 4-tert-octylphenol), or their mixture, covering from 0.5 postcoital day to weaning, continuing in the male offspring each exposure until adulthood (60-days old). Body and relative testis weight were increased in mixture-exposed mice along with histological alterations. Intratesticular testosterone (T) changed only in mice exposed to DBP, whereas estradiol (E2) levels were altered in all groups (except benzyl butyl phthalate). mRNA levels of genes encoding hormones of the steroid pathway (Cyp11a1, Hsd3b1, Cyp17a1, and Cyp19a1), cholesterol transporters (Star), and transcriptional factors (Sp1) showed that mice exposed to single or mixed compounds had alterations in at least 2 transcripts. However, none of the different types of exposure induced changes in all transcripts. In addition, changes at the mRNA or protein levels with single compounds were not always the same as those with a mixture. In conclusion, the effects of a chronic exposure to a mixture of EDCs on the expression of genes and proteins of the steroidogenic pathway and hormonal status were different from those exposed to single EDC.

Endocrine disrupters, microRNAs, and primordial germ cells: a dangerous cocktail

Endocrine-disrupting chemicals (EDCs) are environmental pollutants that may change the homeostasis of the endocrine system, altering the differentiation of germ cells with consequences for reproduction. In mammals, germ cell differentiation begins with primordial germ cells (PGCs) during embryogenesis. Primordial germ cell development and gametogenesis are genetically regulated processes, in which the posttranscriptional gene regulation could be mediated by small noncoding RNAs (sncRNAs) such as microRNAs (miRNAs). Here, we review the deleterious effects of exposure during fetal life to EDCs mediated by deregulation of ncRNAs, and specifically miRNAs on PGC differentiation. Moreover, the environmental stress induced by exposure to some EDCs during the embryonic window of development could trigger reproductive dysfunctions transgenerationally transmitted by epigenetic mechanisms with the involvement of miRNAs expressed in germ line cells.

A human mutated gene is guillotining spermatozoa

At reproductive age, about 13% of the general population suffer from fertility disturbances, with at least a third of them attributable to male factors (1).

An integrative piRNA analysis of mouse gametes and zygotes reveals new potential origins and gene regulatory roles

Piwi-interacting RNAs (piRNAs) are a subclass of the small non-coding RNAs (sncRNAs). Their main reported function was to exert control over transposable elements (TEs) in mammalian germline. In this study undertaking a deeper bioinformatics analysis of piRNAs present in mouse oocytes, sperm cells and zygotes, we first elaborated a new piRNA database based on sequences identified as piRNAs by immunoprecipitation with PIWI proteins. Our bioinformatics analysis revealed that, at least in gametes and zygotes, piRNAs could encompass multifunctional cell-dependent regulatory molecules. Indeed, genome analysis of the piRNA mapping density (reads/kb) evidenced in all samples an enrichment of intron-derived piRNAs. Further, piRNA population was classified into sequences not associated to TEs or repeats (NRapiRNAs) and associated to repetitive genome elements (RapiRNAs). In oocytes most of the NRapiRNAs mapped to the 5′UTRs of coding mRNAs, while higher proportion of NRapiRNAs was detected in sperm cells associated to the 3′UTRs of mRNAs. This piRNA complementarity to mRNA UTRs suggests key post-transcriptional regulatory roles over mRNAs such as those encoding MHC genes. In addition, a striking association of RapiRNA with long non-coding RNAs (lncRNAs) was identified. piRNAs associated with relevant lncRNAs such as: Rab26os and GAS5 and key mRNAs, were particularly assessed.

The landscape of mitochondrial small non-coding RNAs in the PGCs of male mice, spermatogonia, gametes and in zygotes

BackgroundMitochondria are organelles that fulfill a fundamental role in cell bioenergetics, as well as in other processes like cell signaling and death. Small non-coding RNAs (sncRNA) are now being considered as pivotal post-transcriptional regulators, widening the landscape of their diversity and functions. In mammalian cells, small RNAs encoded by the mitochondrial genome, mitosRNAs were discovered recently, although their biological role remains uncertain.ResultsHere, using specific bioinformatics analyses, we have defined the diversity of mitosRNAs present in early differentiated germ cells of male mice (PGCs and spermatogonia), and in the gametes of both sexes and in zygotes. We found strong transcription of mitosRNAs relative to the size of the mtDNA, and classifying these mitosRNAs into different functional sncRNA groups highlighted the predominance of Piwi-interacting RNAs (piRNAs) relative to the other types of mitosRNAs. Mito-piRNAs were more abundant in oocytes and zygotes, where mitochondria fulfill key roles in fecundation process. Functional analysis of some particular mito-piRNAs (mito-piR-7,456,245), also expressed in 3T3-L1 cells, was assessed after exposure to RNA antagonists.ConclusionsAs far as we are aware, this is the first integrated analysis of sncRNAs encoded by mtDNA in germ cells and zygotes. The data obtained suggesting that mitosRNAs fulfill key roles in gamete differentiation and fertilization.

Detection and Characterization of Small Noncoding RNAs in Mouse Gametes and Embryos Prior to Zygotic Genome Activation

Small noncoding RNAs (ncRNAs) are regulatory elements of gene expression in all cell types and tissues. An ever-increasing number of studies have implicated ncRNAs in differentiation and developmental processes. In mammals, as a consequence of fertilization, the content of ncRNAs in the zygote is mostly the result of the maternal material included on oocytes and the potential sperm-borne paternal contributions. The genetic identity program of any individual is the reprogramming of each parental contribution to the zygotic genome activation. In mouse, this activation occurs at 2-cell stage. In this program of early development the small ncRNAs can play important roles. Here, we describe protocols for collection of oocytes, spermatozoa, and zygotes in mouse, followed by RNA purification to analyze the different types of small ncRNA by next-generation sequencing approaches (NGS). Bioinformatics protocols also describe the methodology able to characterize microRNAs (miRNAs) as the most well-known and widespread regulatory small ncRNA. The comparative analysis allows identifying the changes and background previous to zygotic genome activation.