The microbiota and T cells non-genetically modulate inherited phenotypes transgenerationally.

The host-microbiota relationship has evolved to shape mammalian physiology, including immunity, metabolism, and development. Germ-free models are widely used to study microbial effects on host processes such as immunity. Here, we find that both germ-free and T cell-deficient mice exhibit a robust sebum secretion defect persisting across multiple generations despite microbial colonization and T cell repletion. These phenotypes are inherited by progeny conceived during in vitro fertilization using germ-free sperm and eggs, demonstrating that non-genetic information in the gametes is required for microbial-dependent phenotypic transmission. Accordingly, gene expression in early embryos derived from gametes from germ-free or T cell-deficient mice is strikingly and similarly altered. Our findings demonstrate that microbial- and immune-dependent regulation of non-genetic information in the gametes can transmit inherited phenotypes transgenerationally in mice. This mechanism could rapidly generate phenotypic diversity to enhance host adaptation to environmental perturbations.

Extracellular vesicles alter trophoblast function in pregnancies complicated by COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and resulting coronavirus disease (COVID-19) causes placental dysfunction, which increases the risk of adverse pregnancy outcomes. While abnormal placental pathology resulting from COVID-19 is common, direct infection of the placenta is rare. This suggests that pathophysiology associated with maternal COVID-19, rather than direct placental infection, is responsible for placental dysfunction and alteration of the placental transcriptome. We hypothesized that maternal circulating extracellular vesicles (EVs), altered by COVID-19 during pregnancy, contribute to placental dysfunction. To examine this hypothesis, we characterized maternal circulating EVs from pregnancies complicated by COVID-19 and tested their effects on trophoblast cell physiology in vitro . We found that the gestational timing of COVID-19 is a major determinant of circulating EV function and cargo. In vitro trophoblast exposure to EVs isolated from patients with an active infection at the time of delivery, but not EVs isolated from Controls, altered key trophoblast functions including hormone production and invasion. Thus, circulating EVs from participants with an active infection, both symptomatic and asymptomatic cases, can disrupt vital trophoblast functions. EV cargo differed between participants with COVID-19 and Controls, which may contribute to the disruption of the placental transcriptome and morphology. Our findings show that COVID-19 can have effects throughout pregnancy on circulating EVs and circulating EVs are likely to participate in placental dysfunction induced by COVID-19.

A ligation-independent sequencing method reveals tRNA-derived RNAs with blocked 3' termini.

Despite the numerous sequencing methods available, the vast diversity in size and chemical modifications of RNA molecules makes the capture of the full spectrum of cellular RNAs a difficult task. By combining quasi-random hexamer priming with a custom template switching strategy, we developed a method to construct sequencing libraries from RNA molecules of any length and with any type of 3' terminal modification, allowing the sequencing and analysis of virtually all RNA species. Ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to comprehensively characterize changes in small non-coding RNAs and mRNAs simultaneously, with performance comparable to separate dedicated methods. With LIDAR, we comprehensively characterized the coding and non-coding transcriptome of mouse embryonic stem cells, neural progenitor cells, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared to traditional ligation-dependent sequencing methods, and uncovered the presence of tDRs with blocked 3' ends that had previously escaped detection. Our findings highlight the potential of LIDAR to systematically detect all RNAs in a sample and uncover new RNA species with potential regulatory functions.

The microbiota and immune system non-genetically affect offspring phenotypes transgenerationally.

The host-microbiota relationship has evolved to shape mammalian processes, including immunity, metabolism, and development 1-3 . Host phenotypes change in direct response to microbial exposures by the individual. Here we show that the microbiota induces phenotypic change not only in the individual but also in their succeeding generations of progeny. We found that germ-free mice exhibit a robust sebum secretion defect and transcriptional changes in various organs, persisting across multiple generations despite microbial colonization and breeding with conventional mice. Host-microbe interactions could be involved in this process, since T cell-deficient mice, which display defective sebum secretion 4 , also transgenerationally transmit their phenotype to progeny. These phenotypes are inherited by progeny conceived during in vitro fertilization using germ-free sperm and eggs, demonstrating that epigenetic information in the gametes is required for phenotypic transmission. Accordingly, small non-coding RNAs that can regulate embryonic gene expression 5 were strikingly and similarly altered in gametes of germ-free and T cell-deficient mice. Thus, we have uncovered a novel mechanism whereby the microbiota and immune system induce phenotypic changes in successive generations of offspring. This epigenetic form of inheritance could be advantageous for host adaptation to environmental perturbation, where phenotypic diversity can be introduced more rapidly than by genetic mutation.

Cloning and Sequencing Eukaryotic Small RNAs.

Small RNAs are ubiquitous regulators of gene expression that participate in nearly all aspects of physiology in a wide range of organisms. There are many different classes of eukaryotic small RNAs that play regulatory roles at every level of gene expression, including transcription, RNA stability, and translation. While eukaryotic small RNAs display diverse functions across and within classes, they are generally grouped functionally based on the machinery required for their biogenesis, the effector proteins they associate with, and their molecular characteristics. The development of techniques to clone and sequence small RNAs has been critical for their identification, yet the ligation-dependent addition of RNA adapters and the use of reverse transcriptase to generate cDNA in traditional library preparation protocols can be unsuitable to detect certain small RNA subtypes. In particular, 3' or 5' chemical modifications that are characteristic of specific types of small RNAs can impede the ligation-dependent addition of RNA adapters, while internal RNA modifications can interfere with accurate reverse transcription. The inability to clone certain small RNA subtypes with traditional protocols results in an inaccurate assessment of small RNA abundance and diversity, where some RNAs appear over-represented and others are not detected. This overview aims to guide users on how to design small RNA cloning workflows in eukaryotes to more accurately capture specific small RNAs of interest. Hence, we discuss the molecular biology underlying the identification and quantitation of small RNAs, explore the limitations of commonly used protocols, and detail the alternative approaches that can be used to enrich specific small RNA classes. © 2022 Wiley Periodicals LLC.

The Transmission of Intergenerational Epigenetic Information by Sperm microRNAs.

Epigenetic information is transmitted from one generation to the next, modulating the phenotype of offspring non-genetically in organisms ranging from plants to mammals. For intergenerational non-genetic inheritance to occur, epigenetic information must accumulate in germ cells. The three main carriers of epigenetic information-histone post-translational modifications, DNA modifications, and RNAs-all exhibit dynamic patterns of regulation during germ cell development. For example, histone modifications and DNA methylation are extensively reprogrammed and often eliminated during germ cell maturation and after fertilization during embryogenesis. Consequently, much attention has been given to RNAs, specifically small regulatory RNAs, as carriers of inherited epigenetic information. In this review, we discuss examples in which microRNAs have been implicated as key players in transmitting paternal epigenetic information intergenerationally.

Soma-to-germline RNA communication.

More than a century ago, August Weissman defined a distinction between the germline (responsible for propagating heritable information from generation to generation) and the perishable soma. A central motivation for this distinction was to argue against the inheritance of acquired characters, as the germline was partly defined by its protection from external conditions. However, recent decades have seen an explosion of studies documenting the intergenerational and transgenerational effects of environmental conditions, forcing a re-evaluation of how external signals are sensed by, or communicated to, the germline epigenome. Here, motivated by the centrality of small RNAs in paradigms of epigenetic inheritance, we review across species the myriad examples of intercellular RNA trafficking from nurse cells or somatic tissues to developing gametes.

Stability of the cytosine methylome during post-testicular sperm maturation in mouse.

Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotypes. Recent studies show that the small RNA repertoire of sperm is remodeled during post-testicular maturation in the epididymis. Epididymal maturation has also been linked to changes in the sperm methylome, suggesting that the epididymis might play a broader role in shaping the sperm epigenome. Here, we characterize the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. We find very few changes in the cytosine methylation landscape between testicular germ cell populations and cauda epididymal sperm, demonstrating that the sperm methylome is stable throughout post-testicular maturation. Although our sequencing data suggested that caput epididymal sperm exhibit a highly unusual methylome, follow-up studies revealed that this resulted from contamination of caput sperm by extracellular DNA. Extracellular DNA formed web-like structures that ensnared sperm, and was present only in sperm samples obtained from the caput epididymis and vas deferens of virgin males. Curiously, contaminating extracellular DNA was associated with citrullinated histone H3, potentially resulting from a PAD-driven genome decondensation process. Taken together, our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA.

Small RNAs Are Trafficked from the Epididymis to Developing Mammalian Sperm.

The biogenesis of the RNA payload of mature sperm is of great interest, because RNAs delivered to the zygote at fertilization can affect early development. Here, we tested the hypothesis that small RNAs are trafficked to mammalian sperm during the process of post-testicular maturation in the epididymis. By characterizing small RNA dynamics during germ cell maturation in mice, we confirm and extend prior observations that sperm undergo a dramatic switch in the RNA payload from piRNAs to tRNA fragments (tRFs) upon exiting the testis and entering the epididymis. Small RNA delivery to sperm could be recapitulated in vitro by incubating testicular spermatozoa with caput epididymosomes. Finally, tissue-specific metabolic labeling of RNAs in intact mice definitively shows that mature sperm carry RNAs that were originally synthesized in the epididymal epithelium. These data demonstrate that soma-germline RNA transfer occurs in male mammals, most likely via vesicular transport from the epididymis to maturing sperm.

Small RNAs Gained during Epididymal Transit of Sperm Are Essential for Embryonic Development in Mice.

The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) versus distal (cauda) epididymis and then characterized the development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, subsequently implant inefficiently, and fail soon after implantation. Remarkably, microinjection of purified cauda-specific small RNAs into caput-derived embryos not only completely rescued preimplantation molecular defects but also suppressed the post-implantation embryonic lethality phenotype. These findings reveal an essential role for small RNA remodeling during post-testicular maturation of mammalian sperm and identify a specific preimplantation gene expression program responsive to sperm-delivered microRNAs.

Effects of Larval Density on Gene Regulation in Caenorhabditis elegans During Routine L1 Synchronization.

Bleaching gravid C. elegans followed by a short period of starvation of the L1 larvae is a routine method performed by worm researchers for generating synchronous populations for experiments. During the process of investigating dietary effects on gene regulation in L1 stage worms by single-worm RNA-Seq, we found that the density of resuspended L1 larvae affects expression of many mRNAs. Specifically, a number of genes related to metabolism and signaling are highly expressed in worms arrested at low density, but are repressed at higher arrest densities. We generated a GFP reporter strain based on one of the most density-dependent genes in our dataset - lips-15 - and confirmed that this reporter was expressed specifically in worms arrested at relatively low density. Finally, we show that conditioned media from high density L1 cultures was able to downregulate lips-15 even in L1 animals arrested at low density, and experiments using daf-22 mutant animals demonstrated that this effect is not mediated by the ascaroside family of signaling pheromones. Together, our data implicate a soluble signaling molecule in density sensing by L1 stage C. elegans, and provide guidance for design of experiments focused on early developmental gene regulation.

Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.

Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5' fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.

Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans.

During each life cycle, germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute proteins are expressed during male gametogenesis and promote male fertility. Here, we show that the CSR-1 Argonaute functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small RNA signal that represents an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together, these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small RNA complexes that constitute a memory of gene expression in preceding generations.

Functional modularity of nuclear hormone receptors in a Caenorhabditis elegans metabolic gene regulatory network.

Gene regulatory networks (GRNs) provide insights into the mechanisms of differential gene expression at a systems level. GRNs that relate to metazoan development have been studied extensively. However, little is still known about the design principles, organization and functionality of GRNs that control physiological processes such as metabolism, homeostasis and responses to environmental cues. In this study, we report the first experimentally mapped metazoan GRN of Caenorhabditis elegans metabolic genes. This network is enriched for nuclear hormone receptors (NHRs). The NHR family has greatly expanded in nematodes: humans have 48 NHRs, but C. elegans has 284, most of which are uncharacterized. We find that the C. elegans metabolic GRN is highly modular and that two GRN modules predominantly consist of NHRs. Network modularity has been proposed to facilitate a rapid response to different cues. As NHRs are metabolic sensors that are poised to respond to ligands, this suggests that C. elegans GRNs evolved to enable rapid and adaptive responses to different cues by a concurrence of NHR family expansion and modular GRN wiring.

Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans.

Gametogenesis is a thermosensitive process in numerous metazoans, ranging from worms to man. In Caenorhabditis elegans, a variety of RNA-binding proteins that associate with germ-line nuage (P granules), including the Piwi-clade argonaute PRG-1, have been implicated in maintaining fertility at elevated temperature. Here we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3), in promoting thermotolerant male fertility. A rescuing GFP::alg-3 transgene is localized to P granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, the 26G-RNAs which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for thermotolerant fertility in C. elegans and support a model in which AGOs and their small RNA cofactors function to promote robustness in gene-expression networks.