Introduction
The concept of epigenetic inheritance, where ancestral life experiences and environmental factors can influence phenotypes in descendants, is gaining increasing recognition. Parents, particularly fathers, can transmit epigenetic information to their offspring through modifications in their gametes, specifically sperm. This review explores the evidence of across-generational inheritance of paternal environmental effects, focusing on the role of small RNAs (sRNAs) in this process. It delves into recent discoveries regarding the sRNA composition of sperm and how environmental conditions modulate these sRNAs, ultimately affecting offspring phenotypes.
Epigenetic inheritance is defined as the inheritance of phenotypic changes without alterations in the DNA sequence. Parents transmit not only genetic information but also epigenetic information to their offspring. A notable example is genomic imprinting, where gene expression occurs in a parent-of-origin-specific manner. Environmental conditions can also modify epigenetic marks, suggesting that exposure to specific environments can alter gamete epigenetic marks and influence offspring traits.
Intergenerational vs. Transgenerational Inheritance
It is crucial to distinguish between intergenerational and transgenerational inheritance. Intergenerational inheritance involves the transmission of epigenetic information from the F0 generation to the F1 generation in males, as the F0 sperm directly influences the F1 generation. Transgenerational inheritance, on the other hand, involves the inheritance of epigenetic information in the F2 and subsequent generations, as these generations are not directly exposed to the paternal environmental insult. In maternal inheritance, there is direct interaction of the F1 (fetus) and F2 (fetal germ cells) with the maternal environment, making inheritance in F3 and later generations transgenerational.
While the mechanisms behind transgenerational inheritance remain unclear, recent studies have shed light on intergenerational inheritance. The latter is mediated by environment-induced changes in the gamete epigenome, affecting early embryonic development and offspring phenotype. The primary epigenetic information carriers include DNA cytosine methylation, histone modifications, and sRNAs. While the direct role of DNA methylation or histone modifications in intergenerational inheritance is still under investigation, the involvement of sperm sRNAs has been demonstrated in recent years.
The Role of Small RNAs in Epigenetic Inheritance
Types of Small RNAs
A diverse array of sRNAs exists, including well-studied microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), as well as less understood sRNAs like cleavage products from rRNAs and tRNAs. tRNA-derived sRNAs, or tRNA fragments (tRFs), are generated in a site-specific manner from tRNA isotypes. Fragments of rRNAs (rsRNAs) are classified into five types based on their rRNA precursors (5S, 5.8S, 18S, 28S, and 45S). While the function of rsRNAs is unclear, miRNAs, piRNAs, and tRFs play important roles in gene regulation.
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Mechanisms of Action
sRNAs, such as miRNAs and piRNAs, function by binding to Argonaute (Ago) proteins. These sRNA-Ago complexes regulate gene expression post-transcriptionally by degrading or deadenylating target RNA or repressing translation. sRNAs can also regulate gene expression by targeting DNA methylation and chromatin formation at specific genes.
miRNAs are found in various germ cell populations, including mature sperm, with some expressed exclusively in the testes, suggesting a role in spermatogenesis. Studies using Cre drivers for germ-cell-specific deletion of genes involved in miRNA biogenesis have revealed the essential role of miRNAs in germ-cell development and spermatogenesis. Paternal miRNAs and endo-siRNAs are proposed to play an essential role in early embryonic development.
piRNAs, highly expressed in germ cells, bind to the PIWI subfamily of Ago proteins. Two major classes of piRNAs exist in mammals: pre-pachytene piRNAs, which maintain germline genomic integrity by repressing transposon expression, and piRNAs enriched during the pachytene stage of meiosis, which target spermatogenesis-related mRNAs. piRNAs regulate transcription by promoting DNA methylation and post-transcriptionally cleaving target transposon mRNA.
tRFs are abundant in mature sperm, and recent studies have illuminated their biogenesis and functions. These sRNAs include 5′ and 3′ halves of tRNAs generated by cleavage in the anticodon loop and fragments generated via cleavage in the D and T loops of mature or pre-tRNAs. Angiogenin and RNaseT2 endonucleases have been implicated in tRNA processing to generate tRFs, and tRNA modifications potentially regulate their biogenesis and stability. Functionally, tRFs play roles in various cellular processes, including RNA metabolism, translation inhibition, ribosome biogenesis, targeted cleavage of 3′ UTRs, regulation of apoptosis, and regulation of retroviral elements.
Advances in sRNA Sequencing Technologies
Next-generation sequencing (NGS) has revolutionized the discovery and quantification of diverse sRNA classes. However, the structural and chemical modifications on sRNAs can limit their capture in sequencing libraries, depending on the RNA-sequencing protocol used. Traditional ligation-based protocols, which require 5′ phosphate and 3′ hydroxyl groups for adaptor ligation, are most efficient at sequencing miRNAs. However, sRNAs like tRFs and rsRNAs, generated by endonuclease cleavage, may have 2′, 3′ cyclic phosphates or 3′ phosphates, hindering their capture. Additionally, reverse transcriptase (RT) enzymes may struggle to read through modifications on tRNAs, leading to incomplete cDNA synthesis and undetectable sRNAs in the final library.
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Recently developed methods for sRNA sequencing library preparation have addressed some of these issues. ARM-seq and DM-tRNA-seq pretreat RNA with AlkB demethylase to remove modifications like m1A, m3C, and m1G, which interfere with RT. DM-tRNA-seq also uses an engineered mutant of AlkB for more efficient demethylation of m1G. Additional strategies, such as the use of thermostable template-switching RTs like TGIRT, overcome structure and modification-induced RT stops. Ordered Two-Template Relay (OTTR)-seq uses a modified retroelement reverse transcriptase to fuse 5′ and 3′ adaptors during cDNA synthesis, preventing bias introduced during the ligation step. Direct RNA sequencing using Oxford Nanopore Technologies (ONT) allows for the detection of modified nucleotides as part of sequencing, eliminating bias-inducing steps like RT and amplification.
The sRNA Payload of Mature Sperm
With improved sRNA sequencing methods, a more comprehensive picture of the sRNA composition of mature sperm has emerged. In mice, sperm primarily consists of rsRNAs, tRFs, miRNAs, and piRNAs, with rsRNAs and tRFs comprising over 80% of sequencing reads. In human sperm, rsRNAs derived from 28S rRNA are most abundant, accounting for approximately 60% of total rsRNAs. In addition to sRNAs, circular RNAs (circRNAs) have been identified in human and mouse testicular spermatozoa and sperm, functioning as miRNA sponges, protein scaffolds, and translation templates. Furthermore, Isoform Sequencing (Iso-seq) has revealed the long RNA profile of sperm, identifying thousands of full-length intact long RNA transcripts distinct from those in the testes.
Diet-Induced Sperm-Borne Mitochondrial RNAs
Apart from Mendelian inheritance, fathers can transfer intergenerational information through environment-sensitive sncRNAs stored in mature spermatozoa, influencing embryonic development and adult phenotypes. The production of mature spermatozoa involves spermatogenesis and epididymal maturation, both phases constituting potential windows of environmental susceptibility. The sperm sncRNA pool is modified during epididymal transit, with contributions from epididymal epithelial cells.
A study focused on the intergenerational effects of paternal overweight and attempted to dissect the relative contributions of testicular and epididymal exposures. The study found that paternal preconceptional exposure to 2 weeks of a high-fat diet (HFD) at 6 weeks of age, when sperm is undergoing maturation in the epididymis, induces glucose intolerance and insulin resistance in male offspring. The same exposure does not affect the developing germ cells in the testis, indicating that spermatogenesis does not contribute to the paternal intergenerational effects. Mechanistically, mitochondrial-encoded tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) are dynamically regulated by the HFD challenge. These sncRNAs originate from sperm and are enriched in epididymal spermatozoa.
Harnessing the genetic diversity of mtDNA and single-embryo transcriptomics, researchers tracked the parental origin of mt-tRNAs in early embryos and discovered their transfer from spermatozoa at fertilization. The data support a model where acute HFD exposure induces mitochondrial dysfunction in somatic tissues and spermatozoa, compensated by upregulation of mtDNA transcription. This leads to an accumulation of mt-sncRNAs and their fragments, which are epigenetically inherited and modify transcription in early embryos and glucose metabolism in adult offspring. In-depth mouse phenotypic data from the International Mouse Phenotyping Consortium (IMPC) and sperm sncRNA analyses highlighted alterations of spermatozoa mt-tsRNAs downstream of genetically induced mitochondrial dysfunction and paternal non-genetic control of offspring glucose homeostasis. Furthermore, sperm mt-tsRNAs are associated with body mass index (BMI), and paternal BMI at conception is an independent determinant of offspring metabolic health.
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To study the susceptibility of epididymal spermatozoa to diet, male mice were fed HFD or low-fat diet (LFD) for 2 weeks. After the dietary challenge, treated males were either directly mated to generate the F1 generation (eHFD) or allowed to recover on a normal chow diet for 4 weeks before mating (sHFD). Spermatogenesis and male reproductive fitness were not affected by the HFD-feeding, as confirmed by testis histology, sperm motility, and fertilization rates. However, a 2-week HFD exposure led to increased body weight and reduced glucose tolerance in exposed mice.
The HFD challenge in males (eHFD) did not affect offspring body weight but led to glucose intolerance in approximately 30% of male offspring. The glucose intolerance phenotype was robust and stable. These HFDi mice were also significantly insulin resistant. In contrast, offspring of sHFD males showed no alteration in body weight, body composition, or glucose tolerance.
The phenotypic differences between HFDt and HFDi offspring were mirrored by unique transcriptional signatures in metabolically relevant tissues. About 30% of the HFDi signature genes were also expressed in human adipocytes and associated with childhood obesity. In families with lean mothers, paternal overweight doubled offspring obesity risk, worsened by paternal obesity. An increasing paternal BMI was also associated with insulin resistance, with parental BMIs having independent and additive effects on offspring insulin sensitivity.
These results emphasize the importance of paternal preconceptional body weight for offspring metabolic health in mice and humans and also suggests that epididymal spermatozoa can be directly susceptible to environmental cues. sncRNAs in spermatozoa are potential diet-sensitive mediators of paternal epigenetic effects. Profiling sncRNAs from round spermatids and cauda spermatozoa of HFD-fed mice revealed that about 25% of the sperm sncRNA pool is sensitive to the HFD challenge, with predominant reduction of nuclear tRNA expression and fragmentation and upregulation of mt-tRNA expression and fragmentation.
Sperm DNA Methylation and Histone Modification
DNA methylation, which involves the addition of a methyl group to the cytosine ring, plays a crucial role in regulating gene expression, transposon silencing, X chromosome inactivation, and genomic imprinting. DNA methylation is controlled by DNA methyltransferases (DNMTs), while demethylation is orchestrated by Ten-Eleven Translocation enzymes. Methylation patterns in germ cells are established during embryo development, characterized by dynamic de novo methylation, known as DNA reprogramming. Alterations in sperm DNA methylation have been demonstrated to correlate with impaired sperm concentration and motility.
Incomplete erasure of DNA methylation during epigenetic reprogramming may lead to the inheritance of environmental-associated DNA changes to the next generation. Environmental factors can lead to changes in DNA methylation, impacting development. Aberrant DNA methylation in paternally imprinted genes may result in deleterious effects on embryo development.
Unlike somatic cell chromatin, sperm harbor very dense and highly packaged chromatin, where DNA is mostly bound to protamines. During protamination, histones are removed, and the remaining histones are primarily modified by hyperacetylation and butyrylation. These post-translational modifications (PTMs) at H4K5 are associated with alterations in sperm cell genome programming, impacting histone removal during late spermatogenesis.
tags: #epigenetic #inheritance #diet #induced #sperm #borne