piRNAs in sperm function and embryo viability

in Reproduction
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Giulia Perillo Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland

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Keigo Shibata Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland

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Pei-Hsuan Wu Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland

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https://orcid.org/0000-0002-6690-0744

Correspondence should be addressed to P-H Wu; Email: pei-hsuan.wu@unige.ch

*(G Perillo and K Shibata contributed equally to this work)

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In brief

Mouse PIWI-interacting RNAs (piRNAs) are indispensable for spermatogenesis, but whether these small RNAs serve any function beyond gametogenesis is rarely explored. This review summarizes recent findings that demonstrated a requirement for piRNAs in sperm maturation and discusses a potential intergenerational role for paternal piRNAs.

Abstract

Unique to animals, PIWI-interacting RNAs (piRNAs) defend organisms against threats to germline integrity evoked by transposons, retroviruses, and inappropriate expression of protein-coding genes. Characterization of mouse piRNAs and studies of more than a dozen piRNA pathway protein mutants detailed in the past 15 years have firmly established an essential role for piRNAs in male fertility. Despite their vital function in spermatogenesis, mammalian piRNAs were thought to be dispensable beyond gamete formation because all piRNA pathway protein mouse mutants are invariably sterile and do not produce sperm. In contrast to the specialized purpose of piRNAs in gamete formation, tRNA-derived fragments and microRNAs have been the focus of research in RNA-mediated paternal contribution, providing additional examples of the versatility of non-coding RNAs. In recent years, the direct elimination of mouse piRNAs using CRISPR/Cas revealed their extended function in post-testicular sperm maturation. An intergenerational contribution from paternal piRNAs has also been proposed. Together with insights into piRNAs in oocytes and early embryos in mice and other mammals, these newly proposed functions of mammalian piRNAs invite further investigations of piRNA dynamics during sperm maturation and fertilization as well as their roles in reproduction beyond gametogenesis.

Abstract

In brief

Mouse PIWI-interacting RNAs (piRNAs) are indispensable for spermatogenesis, but whether these small RNAs serve any function beyond gametogenesis is rarely explored. This review summarizes recent findings that demonstrated a requirement for piRNAs in sperm maturation and discusses a potential intergenerational role for paternal piRNAs.

Abstract

Unique to animals, PIWI-interacting RNAs (piRNAs) defend organisms against threats to germline integrity evoked by transposons, retroviruses, and inappropriate expression of protein-coding genes. Characterization of mouse piRNAs and studies of more than a dozen piRNA pathway protein mutants detailed in the past 15 years have firmly established an essential role for piRNAs in male fertility. Despite their vital function in spermatogenesis, mammalian piRNAs were thought to be dispensable beyond gamete formation because all piRNA pathway protein mouse mutants are invariably sterile and do not produce sperm. In contrast to the specialized purpose of piRNAs in gamete formation, tRNA-derived fragments and microRNAs have been the focus of research in RNA-mediated paternal contribution, providing additional examples of the versatility of non-coding RNAs. In recent years, the direct elimination of mouse piRNAs using CRISPR/Cas revealed their extended function in post-testicular sperm maturation. An intergenerational contribution from paternal piRNAs has also been proposed. Together with insights into piRNAs in oocytes and early embryos in mice and other mammals, these newly proposed functions of mammalian piRNAs invite further investigations of piRNA dynamics during sperm maturation and fertilization as well as their roles in reproduction beyond gametogenesis.

Introduction

Small RNAs, tiny (19–35 nt) non-coding RNAs that do not produce protein, are universally employed in all kingdoms of life to regulate developmental processes and defend organisms against threats to survival (Iwasaki et al. 2015). PIWI-interacting RNAs (piRNAs; 19–35 nt), microRNAs (miRNAs; ~22 nt), and endogenous small interfering RNAs (endo-siRNAs; ~21 nt) are guardians of germline integrity preserved by evolution. Loaded onto Argonaute family proteins, these small RNAs reinforce appropriate spatial and temporal gene expression and prevent transposon-mediated DNA damage in the animal germline, ensuring fertility and continuation of species.

Animal oocytes are vastly larger and carry more RNA than sperm. For example, human sperm has a cellular volume of ~22 μm3, while a mature metaphase II (MII) oocyte harbors a volume of ~1.8 × 105 μm3 (Curry et al. 1996, Trebichalska et al. 2021). The maternal reservoir of long and small RNAs is indispensable for initiating embryogenesis, a complex process that transforms a fertilized oocyte into an independent multicellular organism (Pauli et al. 2011). By contrast, the contribution of paternal RNAs is not fully understood. The report of Phospholipase C zeta (Plcz1) mRNAs in mouse sperm, which trigger calcium oscillations in oocytes that jumpstart embryogenesis upon sperm entry at fertilization, provided the first example of functional sperm RNAs (Saunders et al. 2002). Since then, accumulating studies proposed an intergenerational role for bulk and specific sperm RNAs (Sharma 2019). In the recent decade, sequencing analyses of sperm RNA and tests of epigenetic inheritance of acquired traits in rodent and invertebrate models have shed new light on paternal small RNAs. Despite their transcriptionally quiescent state, sperm carry diverse classes of small RNAs, including Argonaute-dependent small RNAs and 28–31 nt tRNA-derived fragments, or tRFs (Sharma et al. 2016, Hutcheon et al. 2017, Conine et al. 2018). Studies in worms, flies, mice, and humans have linked sperm tRFs and miRNAs to inheritance of acquired traits resulting from paternal diets and stress (Perez & Lehner 2019, Sharma, 2019). However, mechanistically, how these RNAs achieve regulation in the offspring remains enigmatic.

Among functional paternal small RNAs proposed to date, only piRNAs have become highly specialized in animal fertility in evolution (Iwasaki et al. 2015, Ozata et al. 2019). These 19–35 nt small RNAs display an extraordinary abundance and sequence diversity, distinguished from miRNAs and endo-siRNAs based on a set of well-defined characteristics that include, by definition, their association with the PIWI-clade Argonautes – PIWI proteins. Perplexingly, piRNA sequences diverge rapidly in evolution, but the genomic loci generating piRNAs are conserved in synteny in placental mammals (Ozata et al. 2020). In Drosophila and zebrafish, piRNAs are required for both male and female fertility. In mice, piRNAs are well recognized for their role in spermatogenesis and male fertility. This review highlights recent findings on piRNA functions beyond gamete formation. The defining properties, biogenesis, and functions of piRNAs in spermatogenesis have been extensively reviewed elsewhere (Iwasaki et al. 2015, Ozata et al. 2019) and will be discussed only in the context of the potential intergenerational role of piRNAs. In addition to features shared by all animals, piRNAs have evolved species-specific characteristics. We will focus on mammalian piRNAs, drawing examples from invertebrate models only when applicable.

Fertilization introduces paternal components into oocytes

The paternal contribution of mammalian sperm begins at the fusion of mature gametes via fertilization, a species-specific, stepwise process (Bianchi & Wright 2016). Testicular sperm further mature and gain motility while sequentially transiting through the caput (testis-proximal region), corpus, and cauda (testis-distal region) epididymis (Fig. 1A). In the female reproductive tract, mature sperm become ‘capacitated’ and undergo a series of physical and biochemical transformations that enable them to fertilize oocytes, including the capability to undergo acrosome reaction (Fig. 1B). Acrosome reaction, cellular exocytosis of the sperm acrosome triggered by increased intracellular Ca2+ concentration, releases hydrolytic enzymes and exposes new plasma membrane domains in sperm (Breitbart & Spungin 1997, Ramalho-Santos et al. 2002). On reaching oocytes, the sperm can penetrate the surrounding cumulus cells and bind to the glycoprotein coat enveloping the oocyte plasma membrane called zona pellucida. Subsequently, the sperm fuses with the oocyte plasma membrane, and the whole sperm – including the head and the flagellum – enters the oocyte (Sutovsky et al. 1996, Ramalho-Santos 2011). Once fertilized, the oocyte prevents the entry of a second sperm. Mammalian oocytes achieve such polyspermy block by modifying the plasma membrane, the zona pellucida, or both – depending on the animal species (Evans 2020). The two mechanisms are reminiscent of the ‘fast block’ and ‘slow block’ first defined in externally fertilized marine and aquatic animals. Indeed, mammalian polyspermy block at the zona pellucida through releasing enzymes in cortical granules via exocytosis by the oocyte resembles the long-lasting slow block widely utilized in sexually reproducing animals. However, the rapid and transient depolarization of the oocyte membrane constituting the fast block does not occur in mammals (Evans 2020).

Figure 1
Figure 1

Key events in mammalian fertilization. (A) Maturation of mouse testicular sperm during epididymal transit. The arrow denotes the direction of sperm transport. Small RNA contents in sperm, somatic tissue, and epididymosomes collected from distinct epididymal segments are presented as percentages of total small RNAs reported byHutcheon et al. (2017) and Stanger et al. (2020). Epidsm, epididymosomes. (B) Critical steps in fertilization. In vitro fertilization (IVF) requires all illustrated steps, while intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) can accomplish fertilization even bypassing steps 1 and 2. The oocyte and sperm are not to scale.

Citation: Reproduction 165, 3; 10.1530/REP-22-0312

Gamete fusion triggers oocyte activation, characterized by cytosolic Ca2+ oscillations persisting for hours that is necessary and sufficient to stimulate embryo development (Sanders & Swann 2016). In contrast to the biparental genome, embryos inherit most cytoplasmic molecules – including RNAs and proteins – from the maternal reservoir. Moreover, mammalian mitochondria and mitochondrial DNA are inherited strictly through the female germline, explaining the maternal transmission of mitochondrial diseases. Although sperm mitochondria are delivered to the oocyte during fertilization, they are actively eliminated by ubiquitin-mediated mechanisms in the embryo (Sato & Sato 2013). Similarly, most sperm structural components, such as axoneme and fibrous sheath of the flagellum, are degraded in the zygote. An oddity is the sperm centrosome, which is retained in fertilized oocytes in most mammals – except mice – to promote early embryo division by acting as a microtubule-organizing center that mobilizes and unites the parental pronuclei (Schatten & Sun 2009, Ramalho-Santos 2011). In addition to centrosomes, sperm Plcz1 mRNA and PLCZ1 protein induce intracellular calcium oscillations in oocytes that lead to oocyte activation and the subsequent embryo development (Saunders et al. 2002). No other cytoplasmic paternal factors have been established to influence embryo development. Lastly, the parental pronuclei merge to form the zygotic nucleus, an event more prone to errors in some species than others, possibly due to different centrosome positions and mechanisms of spindle assembly during embryo division (Cavazza et al. 2021).

Fertilization and live births can be achieved in vitro using whole sperm, sperm heads, and even spermatids. In humans, in vitro fertilization (IVF) (Lopata et al. 1980, Jones et al. 1984) and intracytoplasmic sperm injection (ICSI) (Palermo & Van Steirteghem 1991) have revolutionized reproductive medicine. IVF entails incubating capacitated sperm with oocytes, while ICSI can bypass the need for sperm motility by directly injecting a sperm head into an oocyte. Round spermatid injection (ROSI) can also accomplish fertilization and live births (Tanaka et al. 2015), but its application is currently limited to animal research. These methods have proved useful for testing paternal small RNAs. However, because in vitro conditions for fertilization do not fully recapitulate in vivo environments, technical variations in cell manipulation or culture conditions can influence the viability of and epigenetic modifications in embryos (Kohda 2013, Ventura-Junca et al. 2015). For example, independent groups demonstrated conflicting outcomes of mouse ICSI embryos fertilized by caput sperm, which had formed the basis of a potential requirement for specific sperm miRNAs in embryo development (Conine et al. 2018, Fernandez-Gonzalez et al. 2019, Zhou et al. 2019, Wang et al. 2020). The discrepancy was ultimately attributed at least partly to the preparation procedures of sperm heads (Conine et al. 2020, Wang et al. 2020), which underscored the importance of technical details of the IVF method of choice in studies of paternal small RNAs.

Abundance and characteristics of piRNAs in mammalian sperm

Most knowledge of mammalian piRNAs is inferred from studies of mouse piRNAs, which are disproportionally abundant in the male mouse germline and essential only for male fertility. Mouse testes express three types of piRNAs at distinct developmental stages, each associated with one or two of the three mouse PIWI proteins present at the same developmental timing (Fig. 2; Iwasaki et al. 2015, Ozata et al. 2019). Fetal piRNAs (~26 nt and ~28 nt) harbor repetitive sequences and guide MILI (PIWIL2) and MIWI2 (PIWIL4) to silence transposons (Aravin et al. 2007, Aravin et al. 2008, Kuramochi-Miyagawa et al. 2008). Pre-pachytene piRNAs (~26 nt), most plentiful in neonatal mouse testes, are derived from transposons and 3′ untranslated regions of mRNAs and associated with MILI (Aravin et al. 2007, Robine et al. 2009). Pachytene piRNAs (~26 nt and ~30 nt), loaded onto MIWI (PIWIL1) and MILI, begin to accumulate at the pachytene stage of meiosis in spermatogenesis to become the predominant small RNAs in adult testes (>95% of all small RNAs;Aravin et al. 2007, Li et al. 2013). In contrast to transposon-derived piRNAs, most mouse pachytene piRNAs harbor unique sequences produced from 100 genomic regions depleted of protein-coding genes, transposons, and other non-coding RNAs (‘unannotated genomic regions’ in Fig. 2). piRNA-producing loci have also been defined in other mammals (Roovers et al. 2015, Li et al. 2019, Yu et al. 2019, Ozata et al. 2020, Ishino et al. 2021, Loubalova et al. 2021, Zhang et al. 2021).

Figure 2
Figure 2

PIWI proteins and the associated piRNAs in mice and golden hamsters. Four PIWI proteins, present in most mammalian species except mice and rats, are shown. Sequence similarity of full-length mouse (Musmusculus or M.m.), golden hamster (Mesocricetusauratus or M.a.), and human (Homo sapiens or H.s.) PIWI proteins were obtained from UniProt (https://www.uniprot.org/). Dev, development; NA, not applicable; Spc, spermatocyte; Sptd, spermatid

Citation: Reproduction 165, 3; 10.1530/REP-22-0312

Diverse sperm RNA repertoires are consistently observed across mammalian species (Fig. 1A and Table 1). Relative to total sperm small RNAs, piRNAs in rat (29.2%), rabbit (19.1%), and human (11.4%) sperm appear more abundant than mouse sperm (6.5%) (Pantano et al. 2015, Schuster et al. 2016). Sperm are constructed with compartmentalized structures with the head and the flagellum, the latter anatomically further divided into the midpiece, principal piece, and end piece. In mice and rabbits, ~60–80% of total sperm RNA resides in sperm heads (Schuster et al. 2016). Nonetheless, up to ~5-fold enrichment has been reported for piRNAs in mouse sperm flagella, a trend observed also in mature rabbit sperm (Schuster et al. 2016, Sharma et al. 2018). Coincidentally, mouse MIWI and fly Piwi proteins similarly distribute along sperm flagella and localize to a single body adjacent to the sperm nucleus (Hutcheon et al. 2017, Lempradl et al. 2021). In mature fly sperm, expression of other piRNA pathway proteins, such as Aubergine and Ago3, has also been reported (Lempradl et al. 2021). Documented quantities and proportions of sperm RNAs vary even for the same animal species, an observation likely resulting from technical variations in sperm collection and RNA extraction (Mao et al. 2013, Johnson et al. 2015, Schuster et al. 2016, Sharma et al. 2018) or piRNA annotation methods (Table 1). For example, the use of detergent for sperm purification can reduce the sperm RNA yield by as much as 2.6-fold (87 vs 33.6 fg per sperm head) due to loss of the detergent-sensitive outer membrane and extra-nuclear components of sperm (Johnson et al. 2015). Similarly, enriching RNAs ending with 2′-3′ cyclic phosphates or 3′ phosphates with an additional T4 polynucleotide kinase treatment in standard TruSeq small RNA-seq retrieves more tRFs (Sharma et al. 2018).

Table 1

Reported small RNA profiles in mammalian sperm.

Species/strain Source of sperm Sample type Sequencing library construction methods piRNA annotation methods References
Mouse
 C57BL/6J Whole epididymis Whole sperm, head Ion total RNA-seq v2 (Invitrogen) piRNABank (Sai Lakshmi & Agrawal 2008) Schuster et al. 2016
 C57BL/6J Whole epididymis Whole sperm NEXTflex small RNA-seq v3 for Illumina (PerkinElmer) Published piRNA-producing loci (Betel et al. 2007, Rosenkranz 2016) Crisóstomo et al. 2022
 C57BL/6J Whole epididymis Whole sperm Ion total RNA-seq v2 (Invitrogen) NA Yuan et al. 2016
 C57BL/6J Cauda epididymis Whole sperm TruSeq small RNA-seq (Illumina) NA Chen et al. 2016
 FVB/NJ Caput, cauda epididymis Whole sperm TruSeq small RNA-seq (Illumina) Published piRNA-producing loci (Li et al. 2013) Sharma et al. 2016
 FVB/NJ Testis, caput and cauda epididymis Whole sperm, head, flagella TruSeq small RNA-seq (Illumina) Published piRNA-producing loci (Li et al. 2013) Sharma et al. 2018
 SWR/J Caput, corpus, cauda epididymis Whole sperm TruSeq small RNA-seq (Illumina) NA Nixon et al. 2015
 SWR/J Caput, corpus, cauda epididymis Whole sperm TruSeq small RNA-seq (Illumina) proTRAC (Rosenkranz & Zischler 2012) Hutcheon et al. 2017
Rat
Whole epididymis Whole sperm Ion total RNA-seq v2 (Invitrogen) piRNABank (Sai Lakshmi & Agrawal 2008) Schuster et al. 2016
Whole epididymis Whole sperm NEBNext small RNA library prep set for Illumina (NEB) piRNAdb (Piuco & Galante 2021) Suvorov et al. 2020
Boar Ejaculate, caput and cauda epididymis Whole sperm Yang et al. 2016 NA Chen et al. 2020
Rabbit Ejaculate Whole sperm, head Ion total RNA-seq v2 (Invitrogen) piRNABank (Sai Lakshmi & Agrawal 2008) Schuster et al. 2016
Cattle
Ejaculate Whole sperm NEBNext small RNA library prep set for Illumina (NEB) European Nucleotide Archive†; Rosenkranz 2016, Capra et al. 2017 Sellem et al. 2020
Ejaculate Whole sperm TruSeq small RNA-seq (Illumina) proTRAC (Rosenkranz & Zischler 2012) Capra et al. 2017
Caput, cauda epididymis Whole sperm TruSeq small RNA-seq (Illumina) NA Sharma et al. 2016
Human
Ejaculate Whole sperm Ion total RNA-seq v2 (Invitrogen) piRNABank (Sai Lakshmi & Agrawal 2008) Schuster et al. 2016
Ejaculate Whole sperm TruSeq small RNA-seq (Illumina) Girard et al. 2006 Pantano et al. 2015

†Accessible from https://www.ebi.ac.uk/ena/

NA, not applicable (piRNAs not annotated).

Dynamics of sperm piRNAs during epididymal maturation

Consistent with mouse spermatocytes and spermatids, testicular sperm contain abundant piRNAs (>80% of all small RNAs; Sharma et al. 2018). The sperm RNA composition is reshaped by the epididymal transit, which loads sperm with additional tRFs – now the most abundant small RNAs in cauda epididymal sperm – via epididymosomes secreted by epididymal epithelial cells (Sharma et al. 2016, Sharma et al. 2018, Stanger et al. 2020). The transit also alters the sperm miRNA pool, leading to a gain of new miRNA sequences but an overall decrease in bulk miRNAs in cauda epididymal sperm compared to caput epididymal sperm (Hutcheon et al. 2017, Sharma et al. 2018, Trigg et al. 2019). Specific tRFs and miRNAs are associated with sperm epididymal maturation (Nixon et al. 2015, Sharma et al. 2016, Conine et al. 2018, Stanger et al. 2020), indicating that the sperm RNA content does not simply fluctuate. Indeed, the sperm tRFs and miRNAs transferred from epididymides have been proposed to mediate the paternal inheritance of acquired traits and participate in early embryogenesis (Sharma et al. 2016, Conine et al. 2018, Sharma et al. 2018).

Intriguingly, transiting from the testis into caput epididymis, sperm already lose a significant portion of piRNAs (0.2% vs >80% of all small RNAs;Hutcheon et al. 2017, Sharma et al. 2018), suggesting that some piRNAs are eliminated at the beginning of epididymal maturation. piRNAs accumulate again in cauda epididymal sperm (4.9% of all small RNAs;Hutcheon et al. 2017, Sharma et al. 2018). Mouse cauda epididymal sperm piRNAs are ~30 nt long, harbor mostly unique sequences starting with uridine, and originate from intergenic genomic regions, indicating that they are pachytene piRNAs (Hutcheon et al. 2017). Indeed, most identified piRNAs can be traced back to source genomic locations corresponding to pachytene piRNA-producing loci defined in testis (Li et al. 2013). Consistent with this, piRNAs in mature human sperm are ~30 nt long and carry 5′ uridine – features of adult testicular piRNAs (Pantano et al. 2015). Curiously, sequencing of epididymal epithelial cells and epididymosomes detected little piRNA (Sharma et al. 2016, Nixon et al. 2019), excluding epididymides as a major source of the newly appearing piRNAs in cauda epididymal sperm. Supported by MIWI expression in epididymal sperm, it has been postulated that sperm in cauda epididymides produce piRNAs in situ (Hutcheon et al. 2017). However, how and why sperm reacquire piRNAs in cauda epididymides remains a mystery.

In addition to epididymal maturation, altered sperm miRNAs and mRNAs are associated with sperm capacitation in boars (Li et al. 2018), suggesting that capacitation might further modify sperm RNA composition. Together, the data from sperm tRFs, miRNAs, and piRNAs implied that reshaping RNA content may be an integral part of sperm epididymal maturation and capacitation. Although the sperm maturation process and piRNA-producing loci are conserved in placental mammals, sperm piRNA abundance appears to differ across species (Schuster et al. 2016, Ozata et al. 2020). The underlying force behind and the biological relevance of these differences are unclear since the dynamics of piRNAs in sperm maturation have rarely been explored. Clarifying these unknowns has important implications for investigating sperm RNA-mediated paternal inheritance.

piRNAs in mammalian oocytes and embryos

Characterization of maternal and zygotic small RNAs goes hand in hand with studies of paternal RNA-mediated inheritance. Mouse oocytes express MIWI and MILI, but not MIWI2 (Aravin et al. 2008, Ding et al. 2013). MILI-dependent, 26–30 nt piRNAs derived from retrotransposons – mainly long terminal repeat and long interspersed nuclear elements – are found in developing and mature oocytes (Watanabe et al. 2008, Yang et al. 2016). However, in contrast to mouse testis piRNAs, piRNAs in the female mouse germline are far fewer and dispensable (Watanabe et al. 2008, Lim et al. 2013, Yang et al. 2016). Instead, endo-siRNAs produced by an oocyte-specific DICER isoform are the major suppressors of transposons in mouse oogenesis (Watanabe et al. 2008, Flemr et al. 2013, Lim et al. 2013). Consequently, mammalian piRNAs and piRNA pathway proteins were thought futile in females, and mammals seemed inadequate models for exploring piRNA functions in oogenesis. However, accumulating data from non-murine species suggested that piRNA biology in mouse oocytes is unique (Table 2). The best-illustrated examples are piRNAs in golden hamsters (Mesocricetusauratus), members of the Cricetidae family (Fig. 2;Hasuwa et al. 2021, Ishino et al. 2021, Loubalova et al. 2021, Zhang et al. 2021). The Cricetidae and the Muridae, the latter including the widely utilized Musmusculus, diverged about 25 million years ago (Bedford & Hoekstra 2015). Deleting Piwil1 or Mov10l1 – another gene required for piRNA biogenesis – in golden hamsters led to male and female sterility. While the male sterility was readily explained by spermatogenic arrest, Piwil1 –/– and Mov10l1 –/– ovaries developed normally and produced MII oocytes that could be fertilized. However, a lack of maternal PIWIL1 or MOV10L1 proteins in the derived zygotes resulted in arrested embryogenesis at the two-cell stage, accounting for the female sterility phenotype (Hasuwa et al. 2021, Loubalova et al. 2021, Zhang et al. 2021). In comparison, loss of Piwil3, a Piwil1 paralog in golden hamsters, reduced female – but not male – fertility. Similar to fertilized maternal Piwil1-deficient embryos, the reduced female fertility was caused by developmental defects in fertilized maternal Piwil3-deficient embryos (Hasuwa et al. 2021).

Table 2

Published small RNA profiles in mammalian oocytes and embryos.

Cell type/species/strain Developmental stages Sequencing library construction methods piRNA annotation methods References
Oocyte
 Mouse
  C57BL/6J MIIa Ion total RNA-seq v2 (Invitrogen) NAc Yuan et al. 2016
  FVB/N MII Yang et al. 2016 piRNABank (Sai Lakshmi & Agrawal 2008); RNAdb (Pang et al. 2007); Gene Expression Omnibus*; GenBank*; Yang et al. 2016
 Hamster GVa, MII Nextflex small RNA-seq v3 kit (PerkinElmer) Loubalova et al. 2021 Loubalova et al. 2021
 Hamster GV, MII CAS-seq (Yang et al. 2019) Jung et al. 2014 Zhang et al. 2021
 Hamster MII NEXTflex small RNA-seq v3 for Illumina (PerkinElmer) Hasuwa et al. 2021 Hasuwa et al. 2021
 Hamster MII NEBNext small RNA library prep set for Illumina (NEB) Ishino et al. 2021 Ishino et al. 2021
 Bovine GV, MIa, MII NEBNext small RNA library prep set for Illumina (NEB) proTRAC (Rosenkranz & Zischler 2012) Roovers et al. 2015
 Human GV, MI, MII NEBNext small RNA library prep set for Illumina (NEB) Ensembl†; RNAcentral (Petrov et al. 2015); Yang et al. 2019 Paloviita et al. 2021
Embryo
 Mouse
  C57BL/6J 1-cellb–8-cell Ion total RNA-seq v2 (Invitrogen) NA Yuan et al. 2016
  FVB/N 2-cell–blastocyst TruSeq small RNA-seq (Illumina) Published piRNA-producing loci (Li et al. 2013) Sharma et al. 2016
  FVB/N 1-cell–8-cell Yang et al. 2016 piRNABank (Sai Lakshmi & Agrawal 2008); RNAdb (Pang et al. 2007); Gene Expression Omnibus*; GenBank*; Yang et al. 2016
 Hamster 1-cell–4-cell CAS-seq (Yang et al. 2019) Jung et al. 2014 Zhang et al. 2021
 Hamster 2-cell NEXTflex small RNA-seq v3 for Illumina (PerkinElmer) Hasuwa et al. 2021 Hasuwa et al. 2021
 Hamster 2-cell NEBNext small RNA library prep set for Illumina (NEB) Ishino et al. 2021 Ishino et al. 2021
 Bovine 2-cell–4-cell NEBNext small RNA library prep set for Illumina (NEB) proTRAC (Rosenkranz & Zischler 2012) Roovers et al. 2015
 Human 1-cell–8-cell NEBNext small RNA library prep set for Illumina (NEB) Ensembl†; RNAcentral (Petrov et al. 2015); Yang et al. 2019 Paloviita et al. 2021

aGV, germinal vesicle; MI, metaphase I; MII, metaphase II; bOne-cell zygote; cNA, not applicable (piRNAs not annotated); *Accessible at https://www.ncbi.nlm.nih.gov/; †Accessible at http://www.ensembl.org/

Oocyte piRNAs in golden hamsters are loaded onto PIWIL1 or PIWIL3 proteins and display a profile more closely resembling that of human than mouse oocytes (Ishino et al. 2021, Loubalova et al. 2021, Zhang et al. 2021). Two populations of 23–24 nt and 29–30 nt PIWIL1-associated piRNAs, respectively, are present in the oocytes (Ishino et al. 2021, Zhang et al. 2021). In comparison, PIWIL3-associated piRNAs are distinctively shorter (18–20 nt) than other characterized mammalian piRNAs – pre-pachytene and pachytene piRNAs. These short PIWIL3-associated piRNAs also predominate in developing and mature oocytes of primates and humans, in which endo-siRNAs constitute just a minor portion (Yang et al. 2019, Paloviita et al. 2021). Coincidentally, PIWIL3 paralogs are expressed in ovaries in most mammals but not encoded by the mouse or rat genome. Oocytes from several other mammalian species also express more piRNAs than mouse oocytes (Yang et al. 2012, Roovers et al. 2015, Yang et al. 2016), suggesting a more vital function of piRNAs in mammalian oogenesis than previously recognized. Defining and comparing piRNA-producing loci in macaque and bovine ovaries and testes by sequencing piRNAs revealed shared and gamete-specific piRNA-producing genomic loci (Roovers et al. 2015). Unexpectedly, bovine GV, MI, and MII oocyte piRNAs are produced from loci significantly different from those identified based on ovarian piRNAs, even though the piRNA-producing loci remain consistent in oogenesis. The size distributions and types of small RNAs in one-, two-, and even four-cell mouse, bovine, and human embryos mirror those of mature oocytes (Roovers et al. 2015, Yang et al. 2016, Paloviita et al. 2021). In mice, miRNAs synthesized by zygotes begin to accumulate at the two-cell stage while maternal endo-siRNAs and piRNAs gradually diminish (Ohnishi et al. 2010, Yang et al. 2016).

Potential function of mammalian piRNAs beyond spermatogenesis

Recent studies hinted a broader role for piRNAs in sperm maturation and in offspring development. In mice, eliminating pachytene piRNA production from the pi6 locus (6-qF3-28913(−) and 8009(+); chr6:127,753,038–127,818,853 in mm39) left sperm quantity and structure unaltered but impaired capacitation and fertilizing capability of the mutant sperm (Wu et al. 2020). As a result, pi6−/− males were subfertile. In contrast, another mutant that lacked piRNAs from a different major pachytene piRNA-producing locus, pi18 (18-qE-36451.1(−) and 1295(+); chr18:67,162,517–67,217,450 in mm39), displayed a more severe phenotype. pi18−/− males were sterile and produced few sperm with abnormal acrosomes that could not fertilize oocytes (Choi et al. 2021). ICSI or removing the zona pellucida of oocytes in IVF fully bypassed the fertilization defects of pi6−/− sperm, indicating a crucial role for pi6 piRNAs in sperm binding to and penetration of zona pellucida. In comparison, zona pellucida removal partially restored the fertilization rate of pi18−/− sperm to 39.7% – 2.3-fold lower than the control – suggesting that the ensuing events after sperm-and-oocyte binding might also require pi18 piRNAs. Identifying target transcripts of pi6 and pi18 piRNAs by sequencing analyses indicated that they primarily repress mRNAs, not transposons. Transcripts encoding proteins known for their functions in sperm capacitation, including Cation channel sperm-associated auxiliary subunit epsilon 1(Catspere1) mRNAs, were cleaved by pi6 piRNAs in spermatocytes and spermatids (Wu et al. 2020). Dysregulated expression of Golgin subfamily A member 2 (Golga2), which encodes a Golgi matrix protein, was proposed to account for the abnormal acrosome formation in pi18−/− sperm (Choi et al. 2021).

Invertebrate animal models have offered many examples illustrating how maternally inherited piRNAs repress transposons, initiate zygotic piRNA biogenesis in early embryos, and facilitate maternal transcript clearance during the maternal-to-zygotic transition (Senti & Brennecke 2010, Ozata et al. 2019, Ramat & Simonelig 2021). By contrast, an intergenerational role for paternal piRNAs is poorly understood and with few specific examples. In one report, injection of Miwi−/− spermatids into mouse oocytes yielded blastocysts at a rate (27.1%) comparable to wild-type spermatids (25.5%; Yuan et al. 2016). Moreover, after being transferred to surrogate females, Miwi−/− spermatid-derived embryos developed into healthy and fertile offspring at a rate (7.6%) similar to the controls (9.6%), which convincingly argued against an intergenerational role for piRNAs. Nonetheless, the deletion of pi6 piRNAs in mouse sperm led to the developmental arrest of resulting IVF embryos (Wu et al. 2020). Analyses of pi6−/−testicular germ cells and sperm revealed no morphological or structural abnormalities or DNA damage. Moreover, Plcz1 mRNA, a paternal factor crucial for oocyte activation at fertilization, remained unchanged in pi6−/−germ cells (Wu et al. 2020). Together, the results suggested a requirement for paternal piRNAs in early embryo viability. How do we resolve the contradictory observations? Among current IVF methods, ROSI typically leads to a poorer outcome due to a lack of robust oocyte activation (Tao 2022). This common caveat could alter the baseline for standard measurements of developmental success and mask the defects in Miwi−/− spermatid-derived ROSI embryos. Alternatively, although fewer piRNAs are detected in Miwi−/− spermatids, those that persist might serve a unique function in embryos. Characterization of mouse and fly piRNAs showed that most sequences are dispensable, possibly due to the functional redundancy among the vastly diverse piRNA sequences (Xu et al. 2008, Homolka et al. 2015, Wu et al. 2020, Gebert et al. 2021). Indeed, only the topmost abundant sequences present in every germ cell appear functional, while rarer sequences possibly adopt the role of responding to potential threats to the germline (Genzor et al. 2021). Related to this notion, deletion of pi6, pi17 (17-qA3.3–27363(−) and 26735(+); chr17:27,507,249–27,586,457 in mm39;Homolka et al. 2015, Wu et al. 2020), and pi18 piRNAs in mice illustrated that a more prolific piRNA-producing locus does not necessarily correlate with a more severe mouse mutant phenotype. Lastly, in the absence of MIWI, MILI-associated piRNAs might compensate for the loss of MIWI-associated piRNAs (Vourekas et al. 2012, Ding et al. 2018).

Data from a small number of pioneer studies probing the intergenerational role of paternal piRNAs have implicated an influence of paternal diets on the offspring that is linked to piRNAs. In mice, a chronic high-fat diet reduced specific sperm piRNAs in not the exposed animals but their offspring (Crisóstomo et al. 2022). In Drosophila, males with food intake rich in sugar produced embryos expressing altered transcriptome accompanied by a ~10% increase in the whole-body triglyceride level in the adult stage (Lempradl et al. 2021). Complementary to this study is a more detailed depiction of the piRNA pathway in fly spermatogenesis (Quenerch'du et al. 2016, Chen et al. 2021), which has been largely eclipsed by piRNA studies in oogenesis. Like mouse sperm, fly mature sperm carry fewer piRNAs than developing germ cells. Together, these new findings on fly sperm piRNAs can potentially inform paternal piRNA function in other species.

Outstanding questions about paternal piRNAs

Despite growing evidence of their biological significance, the mechanisms of paternally inherited small RNAs remain elusive. Here, we discuss three frequently raised questions in the context of paternal piRNAs. First, how do sperm small RNAs survive acrosomal exocytosis during fertilization? Acrosome reaction in sperm leads to a significant loss of the membrane, the cytoplasm, and likely RNA contained within these structures (Johnson et al. 2015, Leung et al. 2021). Flagella-enriched piRNAs (Hutcheon et al. 2017, Sharma et al. 2018), unexposed to the extracellular environment until the sperm flagellar structure disassembles in the oocyte (Sutovsky et al. 1996, Ramalho-Santos 2011), might be less susceptible to loss during gamete fusion. Alternatively, piRNAs might be retained and stabilized by sperm germ granules analogous to those found in developing germ cells. Although sperm germ granules remain to be identified in most animals, the recent discovery of paternal epigenetic inheritance (PEI) granules in C. elegans (Schreier et al. 2022) raised the curious possibility that such granules might exist in mammals, playing a role in RNA-mediated paternal effects. In C. elegans, PEI granules are specific to sperm and contain a worm Argonaute protein, WAGO3, and PEI proteins that resemble the BTBD domain-containing proteins in humans.

Second, can the inherent properties of small RNA-guided Argonautes give paternal piRNAs an edge in the zygote? Indeed, rich data from multiple groups have established that miRNA-associated Argonautes, such as mouse AGO2, are efficient enzymes capable of catalyzing rapid target cleavage (Salomon et al. 2015, Becker et al. 2019). If piRNA-associated PIWI proteins operate similarly, a small amount of sperm piRNAs can potentially achieve gene regulation in the zygote by executing multiple rounds of target cleavage in a short time. However, recent structural and biochemical interrogations of mouse and sponge (Ephydatiafluviatilis) PIWI proteins showed that PIWI proteins are unexpectedly sluggish enzymes compared to AGO-clade Argonautes. MIWI cleaves its RNA targets 300 times more slowly than mouse AGO2 and remains bound to the cleaved RNA fragments for much longer, leading to slow turnovers (Anzelon et al. 2021, Arif et al. 2022). Together, current evidence suggests that the potential advantage gained by paternal piRNAs more efficiently degrading RNA targets is likely limited in embryos.

Third, if piRNAs cannot rapidly carry out multiple rounds of target cleavage, are paternal piRNAs sufficiently abundant in early embryos to be functional? How do we envision a tiny amount of sperm RNAs (35–65 fg of total RNAs per cell; Schuster et al. 2016) competing with an ocean of maternal RNAs (0.5–1 ng per cell;Boerke et al. 2007)? The >7500-fold difference between paternal and maternal RNAs likely further widens at fertilization, as some sperm RNAs are presumably degraded in the female reproductive tract during sperm transit or in oocytes. Indeed, a quantitative study using a customized sequencing method estimated that a single sperm contributes to just <0.1% of small RNAs in a fertilized mouse oocyte (Yang et al. 2016), a result consistent with the analysis of piRNAs in bovine embryos (Roovers et al. 2015). Small RNAs in mature sperm have been speculated to be modified or preloaded onto effector proteins prior to fertilization (Sharma 2019), which might render them more resistant to degradation by maternal nucleases and efficient in acting on their targets upon entry into oocytes. Supporting this idea, in mature fly sperm, piRNAs are associated with Piwi proteins (Lempradl et al. 2021). In theory, cellular strategies to retain sperm small RNAs and increase their efficiency in embryos – including RNA localization to sperm germ granules and preloading the RNAs onto effector proteins – could be particularly advantageous for the minor population when the parental RNAs are stoichiometrically imbalanced. In this context, although piRNAs in female mice do not fully represent piRNAs in other mammals (Watanabe et al. 2008, Roovers et al. 2015, Yang et al. 2016), the sexually dimorphic piRNA biology in mice might help to probe the stoichiometric relationship between parental piRNAs and its biological consequences in embryogenesis.

Conclusion and perspectives

piRNAs are integral to the formation and post-testicular maturation of mammalian sperm. In addition to regulating sperm function, paternal piRNAs have been proposed to contribute to embryo development, but the experimental evidence remains sparse. Parthenogenetic embryos carrying two maternal genomes usually do not survive due to imbalanced imprinting of the diploid genome and the resulting aberrant expression of imprinted genes. Adjusting the expression of a small number of normally paternally or maternally imprinted genes by deleting one maternal gene copy (Kono et al. 2004) or mimicking imprinting using CRISPR-mediated targeted DNA methylation (Wei et al. 2022) can occasionally overcome this developmental block. Parthenogenetic mice derived solely from unfertilized oocytes with the modified diploid genome develop to adulthood with full vitality and fertility, raising the question of whether epigenetic paternal molecules are necessary. Nonetheless, the efficiency of generating such parthenotes is low (2 out of 457 and 1 out of 227 manipulated oocytes inKono et al. 2004, Wei et al. 2022, respectively), suggesting that additional imprinted genes or cytoplasmic paternal molecules might help promote robust embryogenesis. The results align with the incomplete penetrance of developmental defects observed in pi6–/– sperm-derived embryos (Wu et al. 2020), implying that, if paternal piRNAs serve a function, they may not be absolutely required. Instead, sperm piRNAs might play the role of reducing the inherently stochastic variations in embryo development (Oates 2011), much like how miRNAs confer biological robustness in a wide range of cellular pathways (Ebert & Sharp 2012).

Several limitations must be overcome to gain a deeper understanding of paternal piRNAs. The base pairing rules for piRNAs to find and cleave their targets are not as clearly defined as miRNAs, complicating the identification of piRNA targets in any cell type. Suitable animal models for investigating sperm piRNAs are rare because nearly all published piRNA mutants do not produce sperm. Generating new mutant models by selecting and genetically editing specific piRNAs in vivo is confronted by the challenge of diverse piRNA sequences and the intricately linked biogenesis of piRNAs from the same precursor transcripts (Ozata et al. 2019). A continuously deepening understanding of general properties and mechanisms of piRNAs in diverse animal species will lay the groundwork for grasping how they might operate intergenerationally.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

Funding

This work was supported by the Swiss National Science Foundation (Bern, Switzerland) PRIMA grant (grant number PR00P3_201535).

Author contribution statement

G. P., K. S., and P.-H. W. conceived the ideas, wrote the manuscript, and generated the figures and tables.

References

  • Anzelon TA, Chowdhury S, Hughes SM, Xiao Y, Lander GC & MacRae IJ 2021 Structural basis for piRNA targeting. Nature 597 285289. (https://doi.org/10.1038/s41586-021-03856-x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Aravin AA, Sachidanandam R, Bourc'his D, Schaefer C, Pezic D, Toth KF, Bestor T & Hannon GJ 2008 A piRNA athway primed by individual transposons is linked to de novo DNA methylation in mice. Molecular Cell 31 785799. (https://doi.org/10.1016/j.molcel.2008.09.003)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Aravin AA, Sachidanandam R, Girard A, Fejes-Toth K & Hannon GJ 2007 Developmentally regulated piRNA clusters implicate MILI in transposon control. Science 316 744747. (https://doi.org/10.1126/science.1142612)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Arif A, Bailey S, Izumi N, Anzelon TA, Ozata DM, Andersson C, Gainetdinov I, MacRae IJ, Tomari Y & Zamore PD 2022 GTSF1 accelerates target RNA cleavage by PIWI-clade Argonaute proteins. Nature 608 618625. (https://doi.org/10.1038/s41586-022-05009-0)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Becker WR, Ober-Reynolds B, Jouravleva K, Jolly SM, Zamore PD & Greenleaf WJ 2019 High-throughput analysis reveals rules for target RNA binding and cleavage by AGO2. Molecular Cell 75 741–755.e11. (https://doi.org/10.1016/j.molcel.2019.06.012)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bedford NL & Hoekstra HE 2015 Peromyscus mice as a model for studying natural variation. eLife 4 113. (https://doi.org/10.7554/eLife.06813)

  • Betel D, Sheridan R, Marks DS & Sander C 2007 Computational analysis of mouse piRNA sequence and biogenesis. PLoS Computational Biology 3 e222. (https://doi.org/10.1371/journal.pcbi.0030222)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bianchi E & Wright GJ 2016 Sperm meets egg: the genetics of mammalian fertilization. Annual Review of Genetics 50 93111. (https://doi.org/10.1146/annurev-genet-121415-121834)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Boerke A, Dieleman SJ & Gadella BM 2007 A possible role for sperm RNA in early embryo development. Theriogenology 68(Supplement 1) S147S155. (https://doi.org/10.1016/j.theriogenology.2007.05.058)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Breitbart H & Spungin B 1997 The biochemistry of the acrosome reaction. Molecular Human Reproduction 3 195202. (https://doi.org/10.1093/molehr/3.3.195)

  • Capra E, Turri F, Lazzari B, Cremonesi P, Gliozzi TM, Fojadelli I, Stella A & Pizzi F 2017 Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between high- and low-motile sperm populations. BMC Genomics 18 14. (https://doi.org/10.1186/s12864-016-3394-7)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cavazza T, Takeda Y, Politi AZ, Aushev M, Aldag P, Baker C, Choudhary M, Bucevicius J, Lukinavicius G & Elder K et al.2021 Parental genome unification is highly error-prone in mammalian embryos. Cell 184 28602877.e22. (https://doi.org/10.1016/j.cell.2021.04.013)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen P, Luo Y & Aravin AA 2021 RDC complex executes a dynamic piRNA program during Drosophila spermatogenesis to safeguard male fertility. PLoS Genetics 17 e1009591. (https://doi.org/10.1371/journal.pgen.1009591)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen Q, Yan M, Cao Z, Li X, Zhang Y, Shi J, Feng GH, Peng H, Zhang X & Zhang Y et al.2016 Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder. Science 351 397400. (https://doi.org/10.1126/science.aad7977)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen X, Zheng Y, Lei A, Zhang H, Niu H, Li X, Zhang P, Liao M, Lv Y & Zhu Z et al.2020 Early cleavage of preimplantation embryos is regulated by tRNA(Gln-TTG)-derived small RNAs present in mature spermatozoa. Journal of Biological Chemistry 295 1088510900.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Choi H, Wang Z & Dean J 2021 Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA. PLoS Genetics 17 e1009485. (https://doi.org/10.1371/journal.pgen.1009485)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Conine CC, Sun F, Song L, Rivera-Pérez JA & Rando OJ 2018 Small RNAs gained during epididymal transit of sperm are essential for embryonic development in mice. Developmental Cell 46 470480.e3. (https://doi.org/10.1016/j.devcel.2018.06.024)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Conine CC, Sun F, Song L, Rivera-Perez JA & Rando OJ 2020 Sperm head preparation and genetic background affect caput sperm ICSI embryo viability: cauda-enriched miRNAs only essential in specific conditions. Developmental Cell 55 677678. (https://doi.org/10.1016/j.devcel.2020.11.021)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Crisóstomo L, Bourgery M, Rato L, Raposo JF, Batterham RL, Kotaja N & Alves MG 2022 Testicular “inherited metabolic memory" of ancestral high-fat diet Is associated with sperm sncRNA content. Biomedicines 10 117. (https://doi.org/10.3390/biomedicines10040909)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Curry MR, Millar JD, Tamuli SM & Watson PF 1996 Surface area and volume measurements for ram and human spermatozoa. Biology of Reproduction 55 13251332. (https://doi.org/10.1095/biolreprod55.6.1325)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ding D, Liu J, Midic U, Wu Y, Dong K, Melnick A, Latham KE & Chen C 2018 TDRD5 binds piRNA precursors and selectively enhances pachytene piRNA processing in mice. Nature Communications 9 127. (https://doi.org/10.1038/s41467-017-02622-w)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ding X, Guan H & Li H 2013 Characterization of a piRNA binding protein Miwi in mouse oocytes. Theriogenology 79 610–5.e1 . (https://doi.org/10.1016/j.theriogenology.2012.11.013)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ebert MS & Sharp PA 2012 Roles for microRNAs in conferring robustness to biological processes. Cell 149 515524. (https://doi.org/10.1016/j.cell.2012.04.005)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Evans JP 2020 Preventing polyspermy in mammalian eggs—contributions of the membrane block and other mechanisms. Molecular Reproduction and Development 87 341349. (https://doi.org/10.1002/mrd.23331)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fernandez-Gonzalez R, Laguna R, Ramos-Ibeas P, Pericuesta E, Alcalde-Lopez V, Perez-Cerezales S & Gutierrez-Adan A 2019 Successful ICSI in mice using caput epididymal spermatozoa. Frontiers in Cell and Developmental Biology 7 346. (https://doi.org/10.3389/fcell.2019.00346)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Flemr M, Malik R, Franke V, Nejepinska J, Sedlacek R, Vlahovicek K & Svoboda P 2013 A retrotransposon-driven dicer isoform directs endogenous small interfering RNA production in mouse oocytes. Cell 155 807816. (https://doi.org/10.1016/j.cell.2013.10.001)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gebert D, Neubert LK, Lloyd C, Gui J, Lehmann R & Teixeira FK 2021 Large Drosophila germline piRNA clusters are evolutionarily labile and dispensable for transposon regulation. Molecular Cell 81 39653978.e5. (https://doi.org/10.1016/j.molcel.2021.07.011)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Genzor P, Konstantinidou P, Stoyko D, Manzourolajdad A, Marlin Andrews C, Elchert AR, Stathopoulos C & Haase AD 2021 Cellular abundance shapes function in piRNA-guided genome defense. Genome Research 31 20582068. (https://doi.org/10.1101/gr.275478.121)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Girard A, Sachidanandam R, Hannon GJ & Carmell MA 2006 A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442 199202. (https://doi.org/10.1038/nature04917)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hasuwa H, Iwasaki YW, Au Yeung WK, Ishino K, Masuda H, Sasaki H & Siomi H 2021 Production of functional oocytes requires maternally expressed PIWI genes and piRNAs in golden hamsters. Nature Cell Biology 23 10021012. (https://doi.org/10.1038/s41556-021-00745-3)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Homolka D, Pandey RR, Goriaux C, Brasset E, Vaury C, Sachidanandam R, Fauvarque MO & Pillai RS 2015 PIWI slicing and RNA elements in precursors instruct directional primary piRNA biogenesis. Cell Reports 12 418428. (https://doi.org/10.1016/j.celrep.2015.06.030)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hutcheon K, McLaughlin EA, Stanger SJ, Bernstein IR, Dun MD, Eamens AL & Nixon B 2017 Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa. RNA Biology 14 17761790. (https://doi.org/10.1080/15476286.2017.1356569)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ishino K, Hasuwa H, Yoshimura J, Iwasaki YW, Nishihara H, Seki NM, Hirano T, Tsuchiya M, Ishizaki H & Masuda H et al.2021 Hamster PIWI proteins bind to piRNAs with stage-specific size variations during oocyte maturation. Nucleic Acids Research 49 27002720. (https://doi.org/10.1093/nar/gkab059)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Iwasaki YW, Siomi MC & Siomi H 2015 PIWI-interacting RNA: its biogenesis and functions. Annual Review of Biochemistry 84 405433. (https://doi.org/10.1146/annurev-biochem-060614-034258)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Johnson GD, Mackie P, Jodar M, Moskovtsev S & Krawetz SA 2015 Chromatin and extracellular vesicle associated sperm RNAs. Nucleic Acids Research 43 68476859. (https://doi.org/10.1093/nar/gkv591)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Jones HW Jr, Acosta AA, Andrews MC, Garcia JE, Jones GS, Mayer J, McDowell JS, Rosenwaks Z, Sandow BA & Veeck LL 1984 Three years of in vitro fertilization at Norfolk. Fertility and Sterility 42 826834. (https://doi.org/10.1016/s0015-0282(1648251-8)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Jung I, Park JC & Kim S 2014 piClust: a density based piRNA clustering algorithm. Computational Biology and Chemistry 50 6067. (https://doi.org/10.1016/j.compbiolchem.2014.01.008)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kohda T 2013 Effects of embryonic manipulation and epigenetics. Journal of Human Genetics 58 416420. (https://doi.org/10.1038/jhg.2013.61)

  • Kono T, Obata Y, Wu Q, Niwa K, Ono Y, Yamamoto Y, Park ES, Seo JS & Ogawa H 2004 Birth of parthenogenetic mice that can develop to adulthood. Nature 428 860864. (https://doi.org/10.1038/nature02402)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kuramochi-Miyagawa S, Watanabe T, Gotoh K, Totoki Y, Toyoda A, Ikawa M, Asada N, Kojima K, Yamaguchi Y & Ijiri TW et al.2008 DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. Genes and Development 22 908917. (https://doi.org/10.1101/gad.1640708)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lempradl A, Kugelberg U, Iconomou M, Beddows I, Nätt D, Casas E, Örkenby L, Enders L, Gutierrez Martinez A & Boonen E et al.2021 Intergenerational metabolic priming by sperm piRNAs. bioRxiv. (https://doi.org/10.1101/2021.03.29.436592)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Leung MR, Ravi RT, Gadella BM & Zeev-Ben-Mordehai T 2021 Membrane remodeling and matrix dispersal intermediates during mammalian acrosomal exocytosis. Frontiers in Cell and Developmental Biology 9 765673. (https://doi.org/10.3389/fcell.2021.765673)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li B, He X, Zhao Y, Bai D, Bou G, Zhang X, Su S, Dao L, Liu R & Wang Y et al.2019 Identification of piRNAs and piRNA clusters in the testes of the Mongolian Horse. Scientific Reports 9 5022. (https://doi.org/10.1038/s41598-019-41475-9)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li XZ, Roy CK, Dong X, Bolcun-Filas E, Wang J, Han BW, Xu J, Moore MJ, Schimenti JC & Weng Z et al.2013 An ancient transcription factor initiates the burst of piRNA production during early meiosis in mouse testes. Molecular Cell 50 6781. (https://doi.org/10.1016/j.molcel.2013.02.016)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li Y, Li RH, Ran MX, Zhang Y, Liang K, Ren YN, He WC, Zhang M, Zhou GB & Qazi IH et al.2018 High throughput small RNA and transcriptome sequencing reveal capacitation-related microRNAs and mRNA in boar sperm. BMC Genomics 19 736. (https://doi.org/10.1186/s12864-018-5132-9)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lim AK, Lorthongpanich C, Chew TG, Tan CW, Shue YT, Balu S, Gounko N, Kuramochi-Miyagawa S, Matzuk MM & Chuma S et al.2013 The nuage mediates retrotransposon silencing in mouse primordial ovarian follicles. Development 140 38193825. (https://doi.org/10.1242/dev.099184)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lopata A, Johnston IW, Hoult IJ & Speirs AI 1980 Pregnancy following intrauterine implantation of an embryo obtained by in vitro fertilization of a preovulatory egg. Fertility and Sterility 33 117120. (https://doi.org/10.1016/s0015-0282(1644529-2)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Loubalova Z, Fulka H, Horvat F, Pasulka J, Malik R, Hirose M, Ogura A & Svoboda P 2021 Formation of spermatogonia and fertile oocytes in golden hamsters requires piRNAs. Nature Cell Biology 23 9921001. (https://doi.org/10.1038/s41556-021-00746-2)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mao S, Goodrich RJ, Hauser R, Schrader SM, Chen Z & Krawetz SA 2013 Evaluation of the effectiveness of semen storage and sperm purification methods for spermatozoa transcript profiling. Systems Biology in Reproductive Medicine 59 287295. (https://doi.org/10.3109/19396368.2013.817626)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Nixon B, De Iuliis GN, Dun MD, Zhou W, Trigg NA & Eamens AL 2019 Profiling of epididymal small non-protein-coding RNAs. Andrology 7 669680. (https://doi.org/10.1111/andr.12640)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Nixon B, Stanger SJ, Mihalas BP, Reilly JN, Anderson AL, Tyagi S, Holt JE & McLaughlin EA 2015 The microRNA signature of mouse spermatozoa is substantially modified during epididymal maturation. Biology of Reproduction 93 91. (https://doi.org/10.1095/biolreprod.115.132209)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Oates AC 2011 What's all the noise about developmental stochasticity? Development 138 601607. (https://doi.org/10.1242/dev.059923)

  • Ohnishi Y, Totoki Y, Toyoda A, Watanabe T, Yamamoto Y, Tokunaga K, Sakaki Y, Sasaki H & Hohjoh H 2010 Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development. Nucleic Acids Research 38 51415151. (https://doi.org/10.1093/nar/gkq229)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ozata DM, Gainetdinov I, Zoch A, O’Carroll D & Zamore PD 2019 PIWI-interacting RNAs: small RNAs with big functions. Nature Reviews. Genetics 20 89108. (https://doi.org/10.1038/s41576-018-0073-3)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ozata DM, Yu T, Mou H, Gainetdinov I, Colpan C, Cecchini K, Kaymaz Y, Wu PH, Fan K & Kucukural A et al.2020 Evolutionarily conserved pachytene piRNA loci are highly divergent among modern humans. Nature Ecology and Evolution 4 156168. (https://doi.org/10.1038/s41559-019-1065-1)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Palermo G & Van Steirteghem A 1991 Enhancement of acrosome reaction and subzonal insemination of a single spermatozoon in mouse eggs. Molecular Reproduction and Development 30 339345. (https://doi.org/10.1002/mrd.1080300408)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Paloviita P, Hyden-Granskog C, Yohannes DA, Paluoja P, Kere J, Tapanainen JS, Krjutskov K, Tuuri T, Vosa U & Vuoristo S 2021 Small RNA expression and miRNA modification dynamics in human oocytes and early embryos. Genome Research 31 14741485. (https://doi.org/10.1101/gr.268193.120)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pang KC, Stephen S, Dinger ME, Engstrom PG, Lenhard B & Mattick JS 2007 RNAdb 2.0—an expanded database of mammalian non-coding RNAs. Nucleic Acids Research 35 D178D182. (https://doi.org/10.1093/nar/gkl926)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pantano L, Jodar M, Bak M, Ballesca JL, Tommerup N, Oliva R & Vavouri T 2015 The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes. RNA 21 10851095. (https://doi.org/10.1261/rna.046482.114)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pauli A, Rinn JL & Schier AF 2011 Non-coding RNAs as regulators of embryogenesis. Nature Reviews. Genetics 12 136149. (https://doi.org/10.1038/nrg2904)

  • Perez MF & Lehner B 2019 Intergenerational and transgenerational epigenetic inheritance in animals. Nature Cell Biology 21 143151. (https://doi.org/10.1038/s41556-018-0242-9)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Piuco R & Galante PAF 2021 piRNAdb: A Piwi-Interacting RNA Database . bioRxiv 09.21.461238. (https://doi.org/10.1101/2021.09.21.461238)

  • Quenerch'du E, Anand A & Kai T 2016 The piRNA pathway is developmentally regulated during spermatogenesis in Drosophila. RNA 22 10441054. (https://doi.org/10.1261/rna.055996.116)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ramalho-Santos J 2011 A sperm's tail: the importance of getting it right. Human Reproduction 26 25902591. (https://doi.org/10.1093/humrep/der200)

  • Ramalho-Santos J, Schatten G & Moreno RD 2002 Control of membrane fusion during spermiogenesis and the acrosome reaction. Biology of Reproduction 67 10431051. (https://doi.org/10.1095/biolreprod67.4.1043)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ramat A & Simonelig M 2021 Functions of PIWI proteins in gene regulation: new arrows added to the piRNA quiver. Trends in Genetics 37 188200. (https://doi.org/10.1016/j.tig.2020.08.011)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • RNAcentral Consortium, Petrov AI, Kay SJE, Gibson R, Kulesha E, Staines D, Bruford EA, Wright MW, Burge S & Finn RD et al.2015 RNAcentral: an international database of ncRNA sequences. Nucleic Acids Research 43 D123D129. (https://doi.org/10.1093/nar/gku991)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Robine N, Lau NC, Balla S, Jin Z, Okamura K, Kuramochi-Miyagawa S, Blower MD & Lai EC 2009 A broadly conserved pathway generates 3'UTR-directed primary piRNAs. Current Biology 19 20662076. (https://doi.org/10.1016/j.cub.2009.11.064)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Roovers EF, Rosenkranz D, Mahdipour M, Han CT, He N, Chuva de Sousa Lopes SM, van der Westerlaken LA, Zischler H, Butter F & Roelen BA et al.2015 Piwi proteins and piRNAs in mammalian oocytes and early embryos. Cell Reports 10 20692082. (https://doi.org/10.1016/j.celrep.2015.02.062)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rosenkranz D 2016 piRNA cluster database: a web resource for piRNA producing loci. Nucleic Acids Research 44 D223D230. (https://doi.org/10.1093/nar/gkv1265)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rosenkranz D & Zischler H 2012 proTRAC - a software for probabilistic piRNA cluster detection, visualization and analysis. BMC Bioinformatics 13 5. (https://doi.org/10.1186/1471-2105-13-5)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sai Lakshmi S & Agrawal S 2008 piRNABank: a web resource on classified and clustered piwi-interacting RNAs. Nucleic Acids Research 36 D173D177. (https://doi.org/10.1093/nar/gkm696)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Salomon WE, Jolly SM, Moore MJ, Zamore PD & Serebrov V 2015 Single-molecule imaging reveals that Argonaute reshapes the binding properties of its nucleic acid guides. Cell 162 8495. (https://doi.org/10.1016/j.cell.2015.06.029)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sanders JR & Swann K 2016 Molecular triggers of egg activation at fertilization in mammals. Reproduction 152 R41R50. (https://doi.org/10.1530/REP-16-0123)

  • Sato M & Sato K 2013 Maternal inheritance of mitochondrial DNA by diverse mechanisms to eliminate paternal mitochondrial DNA. Biochimica et Biophysica Acta 1833 19791984. (https://doi.org/10.1016/j.bbamcr.2013.03.010)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann K & Lai FA 2002 PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development. Development 129 35333544. (https://doi.org/10.1242/dev.129.15.3533)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Schatten H & Sun QY 2009 The role of centrosomes in mammalian fertilization and its significance for ICSI. Molecular Human Reproduction 15 531538. (https://doi.org/10.1093/molehr/gap049)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Schreier J, Dietz S, Boermel M, Oorschot V, Seistrup AS, de Jesus Domingues AM, Bronkhorst AW, Nguyen DAH, Phillis S & Gleason EJ et al.2022 Membrane-associated cytoplasmic granules carrying the Argonaute protein WAGO-3 enable paternal epigenetic inheritance in Caenorhabditis elegans. Nature Cell Biology 24 217229. (https://doi.org/10.1038/s41556-021-00827-2)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Schuster A, Tang C, Xie Y, Ortogero N, Yuan S & Yan W 2016 SpermBase: a database for sperm-borne RNA contents. Biology of Reproduction 95 99. (https://doi.org/10.1095/biolreprod.116.142190)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sellem E, Marthey S, Rau A, Jouneau L, Bonnet A, Perrier JP, Fritz S, Le Danvic C, Boussaha M & Kiefer H et al.2020 A comprehensive overview of bull sperm-borne small non-coding RNAs and their diversity across breeds. Epigenetics and Chromatin 13 19. (https://doi.org/10.1186/s13072-020-00340-0)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Senti KA & Brennecke J 2010 The piRNA pathway: a fly's perspective on the guardian of the genome. Trends in Genetics 26 499509. (https://doi.org/10.1016/j.tig.2010.08.007)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sharma U 2019 Paternal contributions to offspring health: role of sperm small RNAs in intergenerational transmission of epigenetic information. Frontiers in Cell and Developmental Biology 7 215. (https://doi.org/10.3389/fcell.2019.00215)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sharma U, Conine CC, Shea JM, Boskovic A, Derr AG, Bing XY, Belleannee C, Kucukural A, Serra RW & Sun F et al.2016 Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals. Science 351 391396. (https://doi.org/10.1126/science.aad6780)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sharma U, Sun F, Conine CC, Reichholf B, Kukreja S, Herzog VA, Ameres SL & Rando OJ 2018 Small RNAs are trafficked from the epididymis to developing mammalian sperm. Developmental Cell 46 481–494.e6. (https://doi.org/10.1016/j.devcel.2018.06.023)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Stanger SJ, Bernstein IR, Anderson AL, Hutcheon K, Dun MD, Eamens AL & Nixon B 2020 The abundance of a transfer RNA-derived RNA fragment small RNA subpopulation is enriched in cauda spermatozoa. ExRNA 2 17. (https://doi.org/10.1186/s41544-020-00058-x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sutovsky P, Navara CS & Schatten G 1996 Fate of the sperm mitochondria, and the incorporation, conversion, and disassembly of the sperm tail structures during bovine fertilization. Biology of Reproduction 55 11951205. (https://doi.org/10.1095/biolreprod55.6.1195)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Suvorov A, Pilsner JR, Naumov V, Shtratnikova V, Zheludkevich A, Gerasimov E, Logacheva M & Sergeyev O 2020 Aging induces profound changes in sncRNA in rat sperm and these changes are modified by perinatal exposure to environmental flame retardant. International Journal of Molecular Sciences 21 8252. (https://doi.org/10.3390/ijms21218252)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tanaka A, Nagayoshi M, Takemoto Y, Tanaka I, Kusunoki H, Watanabe S, Kuroda K, Takeda S, Ito M & Yanagimachi R 2015 Fourteen babies born after round spermatid injection into human oocytes. Proceedings of the National Academy of Sciences of the United States of America 112 1462914634. (https://doi.org/10.1073/pnas.1517466112)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tao Y 2022 Oocyte activation during round spermatid injection: state of the art. Reproductive Biomedicine Online 45 211218. (https://doi.org/10.1016/j.rbmo.2022.03.024)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Trebichalska Z, Javurek J, Tatickova M, Kyjovska D, Kloudova S, Otevrel P, Hampl A & Holubcova Z 2021 High-resolution 3D reconstruction of human oocytes using focused ion beam scanning electron microscopy. Frontiers in Cell and Developmental Biology 9 755740. (https://doi.org/10.3389/fcell.2021.755740)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Trigg NA, Eamens AL & Nixon B 2019 The contribution of epididymosomes to the sperm small RNA profile. Reproduction 157 R209R223. (https://doi.org/10.1530/REP-18-0480)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ventura-Junca P, Irarrazaval I, Rolle AJ, Gutierrez JI, Moreno RD & Santos MJ 2015 In vitro fertilization (IVF) in mammals: epigenetic and developmental alterations. Scientific and bioethical implications for IVF in humans. Biological Research 48 68. (https://doi.org/10.1186/s40659-015-0059-y)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Vourekas A, Zheng Q, Alexiou P, Maragkakis M, Kirino Y, Gregory BD & Mourelatos Z 2012 Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis. Nature Structural and Molecular Biology 19 773781. (https://doi.org/10.1038/nsmb.2347)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Wang Y, Yamauchi Y, Wang Z, Zheng H, Yanagimachi R, Ward MA & Yan W 2020 Both cauda and caput epididymal sperm are capable of supporting full-term development in FVB and CD-1 mice. Developmental Cell 55 675676. (https://doi.org/10.1016/j.devcel.2020.11.022)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, Obata Y, Chiba H, Kohara Y, Kono T & Nakano T et al.2008 Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453 539543. (https://doi.org/10.1038/nature06908)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Wei Y, Yang CR & Zhao ZA 2022 Viable offspring derived from single unfertilized mammalian oocytes. Proceedings of the National Academy of Sciences of the United States of America 119 e2115248119. (https://doi.org/10.1073/pnas.2115248119)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Wu PH, Fu Y, Cecchini K, Özata DM, Arif A, Yu T, Colpan C, Gainetdinov I, Weng Z & Zamore PD 2020 The evolutionarily conserved piRNA-producing locus pi6 is required for male mouse fertility. Nature Genetics 52 728739. (https://doi.org/10.1038/s41588-020-0657-7)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Xu M, You Y, Hunsicker P, Hori T, Small C, Griswold MD & Hecht NB 2008 Mice deficient for a small cluster of piwi-interacting RNAs implicate piwi-interacting RNAs in transposon control. Biology of Reproduction 79 5157. (https://doi.org/10.1095/biolreprod.108.068072)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yang CX, Du ZQ, Wright EC, Rothschild MF, Prather RS & Ross JW 2012 Small RNA profile of the cumulus-oocyte complex and early embryos in the pig. Biology of Reproduction 87 117. (https://doi.org/10.1095/biolreprod.111.096669)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yang Q, Li R, Lyu Q, Hou L, Liu Z, Sun Q, Liu M, Shi H, Xu B & Yin M et al.2019 Single-cell CAS-seq reveals a class of short PIWI-interacting RNAs in human oocytes. Nature Communications 10 3389. (https://doi.org/10.1038/s41467-019-11312-8)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yang Q, Lin J, Liu M, Li R, Tian B, Zhang X, Xu B, Liu M, Zhang X & Li Y et al.2016 Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos. Science Advances 2 e1501482. (https://doi.org/10.1126/sciadv.1501482)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yu T, Koppetsch BS, Pagliarani S, Johnston S, Silverstein NJ, Luban J, Chappell K, Weng Z & Theurkauf WE 2019 The piRNA response to retroviral invasion of the koala genome. Cell 179 632.e612643. e612. (https://doi.org/10.1016/j.cell.2019.09.002)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yuan S, Schuster A, Tang C, Yu T, Ortogero N, Bao J, Zheng H & Yan W 2016 Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development. Development 143 635647. (https://doi.org/10.1242/dev.131755)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yuan S, Tang C, Schuster A, Zhang Y, Zheng H & Yan W 2016 Paternal pachytene piRNAs are not required for fertilization, embryonic development and sperm-mediated epigenetic inheritance in mice. Environmental Epigenetics 2 dvw021. (https://doi.org/10.1093/eep/dvw021)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhang H, Zhang F, Chen Q, Li M, Lv X, Xiao Y, Zhang Z, Hou L, Lai Y & Zhang Y et al.2021 The piRNA pathway is essential for generating functional oocytes in golden hamsters. Nature Cell Biology 23 10131022. (https://doi.org/10.1038/s41556-021-00750-6)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhou D, Suzuki T, Asami M & Perry ACF 2019 Caput epididymidal mouse sperm support full development. Developmental Cell 50 56. (https://doi.org/10.1016/j.devcel.2019.05.012)

    • PubMed
    • Search Google Scholar
    • Export Citation

 

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  • Figure 1

    Key events in mammalian fertilization. (A) Maturation of mouse testicular sperm during epididymal transit. The arrow denotes the direction of sperm transport. Small RNA contents in sperm, somatic tissue, and epididymosomes collected from distinct epididymal segments are presented as percentages of total small RNAs reported byHutcheon et al. (2017) and Stanger et al. (2020). Epidsm, epididymosomes. (B) Critical steps in fertilization. In vitro fertilization (IVF) requires all illustrated steps, while intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) can accomplish fertilization even bypassing steps 1 and 2. The oocyte and sperm are not to scale.

  • Figure 2

    PIWI proteins and the associated piRNAs in mice and golden hamsters. Four PIWI proteins, present in most mammalian species except mice and rats, are shown. Sequence similarity of full-length mouse (Musmusculus or M.m.), golden hamster (Mesocricetusauratus or M.a.), and human (Homo sapiens or H.s.) PIWI proteins were obtained from UniProt (https://www.uniprot.org/). Dev, development; NA, not applicable; Spc, spermatocyte; Sptd, spermatid

  • Anzelon TA, Chowdhury S, Hughes SM, Xiao Y, Lander GC & MacRae IJ 2021 Structural basis for piRNA targeting. Nature 597 285289. (https://doi.org/10.1038/s41586-021-03856-x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Aravin AA, Sachidanandam R, Bourc'his D, Schaefer C, Pezic D, Toth KF, Bestor T & Hannon GJ 2008 A piRNA athway primed by individual transposons is linked to de novo DNA methylation in mice. Molecular Cell 31 785799. (https://doi.org/10.1016/j.molcel.2008.09.003)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Aravin AA, Sachidanandam R, Girard A, Fejes-Toth K & Hannon GJ 2007 Developmentally regulated piRNA clusters implicate MILI in transposon control. Science 316 744747. (https://doi.org/10.1126/science.1142612)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Arif A, Bailey S, Izumi N, Anzelon TA, Ozata DM, Andersson C, Gainetdinov I, MacRae IJ, Tomari Y & Zamore PD 2022 GTSF1 accelerates target RNA cleavage by PIWI-clade Argonaute proteins. Nature 608 618625. (https://doi.org/10.1038/s41586-022-05009-0)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Becker WR, Ober-Reynolds B, Jouravleva K, Jolly SM, Zamore PD & Greenleaf WJ 2019 High-throughput analysis reveals rules for target RNA binding and cleavage by AGO2. Molecular Cell 75 741–755.e11. (https://doi.org/10.1016/j.molcel.2019.06.012)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bedford NL & Hoekstra HE 2015 Peromyscus mice as a model for studying natural variation. eLife 4 113. (https://doi.org/10.7554/eLife.06813)

  • Betel D, Sheridan R, Marks DS & Sander C 2007 Computational analysis of mouse piRNA sequence and biogenesis. PLoS Computational Biology 3 e222. (https://doi.org/10.1371/journal.pcbi.0030222)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bianchi E & Wright GJ 2016 Sperm meets egg: the genetics of mammalian fertilization. Annual Review of Genetics 50 93111. (https://doi.org/10.1146/annurev-genet-121415-121834)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Boerke A, Dieleman SJ & Gadella BM 2007 A possible role for sperm RNA in early embryo development. Theriogenology 68(Supplement 1) S147S155. (https://doi.org/10.1016/j.theriogenology.2007.05.058)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Breitbart H & Spungin B 1997 The biochemistry of the acrosome reaction. Molecular Human Reproduction 3 195202. (https://doi.org/10.1093/molehr/3.3.195)

  • Capra E, Turri F, Lazzari B, Cremonesi P, Gliozzi TM, Fojadelli I, Stella A & Pizzi F 2017 Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between high- and low-motile sperm populations. BMC Genomics 18 14. (https://doi.org/10.1186/s12864-016-3394-7)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cavazza T, Takeda Y, Politi AZ, Aushev M, Aldag P, Baker C, Choudhary M, Bucevicius J, Lukinavicius G & Elder K et al.2021 Parental genome unification is highly error-prone in mammalian embryos. Cell 184 28602877.e22. (https://doi.org/10.1016/j.cell.2021.04.013)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen P, Luo Y & Aravin AA 2021 RDC complex executes a dynamic piRNA program during Drosophila spermatogenesis to safeguard male fertility. PLoS Genetics 17 e1009591. (https://doi.org/10.1371/journal.pgen.1009591)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen Q, Yan M, Cao Z, Li X, Zhang Y, Shi J, Feng GH, Peng H, Zhang X & Zhang Y et al.2016 Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder. Science 351 397400. (https://doi.org/10.1126/science.aad7977)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen X, Zheng Y, Lei A, Zhang H, Niu H, Li X, Zhang P, Liao M, Lv Y & Zhu Z et al.2020 Early cleavage of preimplantation embryos is regulated by tRNA(Gln-TTG)-derived small RNAs present in mature spermatozoa. Journal of Biological Chemistry 295 1088510900.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Choi H, Wang Z & Dean J 2021 Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA. PLoS Genetics 17 e1009485. (https://doi.org/10.1371/journal.pgen.1009485)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Conine CC, Sun F, Song L, Rivera-Pérez JA & Rando OJ 2018 Small RNAs gained during epididymal transit of sperm are essential for embryonic development in mice. Developmental Cell 46 470480.e3. (https://doi.org/10.1016/j.devcel.2018.06.024)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Conine CC, Sun F, Song L, Rivera-Perez JA & Rando OJ 2020 Sperm head preparation and genetic background affect caput sperm ICSI embryo viability: cauda-enriched miRNAs only essential in specific conditions. Developmental Cell 55 677678. (https://doi.org/10.1016/j.devcel.2020.11.021)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Crisóstomo L, Bourgery M, Rato L, Raposo JF, Batterham RL, Kotaja N & Alves MG 2022 Testicular “inherited metabolic memory" of ancestral high-fat diet Is associated with sperm sncRNA content. Biomedicines 10 117. (https://doi.org/10.3390/biomedicines10040909)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Curry MR, Millar JD, Tamuli SM & Watson PF 1996 Surface area and volume measurements for ram and human spermatozoa. Biology of Reproduction 55 13251332. (https://doi.org/10.1095/biolreprod55.6.1325)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ding D, Liu J, Midic U, Wu Y, Dong K, Melnick A, Latham KE & Chen C 2018 TDRD5 binds piRNA precursors and selectively enhances pachytene piRNA processing in mice. Nature Communications 9 127. (https://doi.org/10.1038/s41467-017-02622-w)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ding X, Guan H & Li H 2013 Characterization of a piRNA binding protein Miwi in mouse oocytes. Theriogenology 79 610–5.e1 . (https://doi.org/10.1016/j.theriogenology.2012.11.013)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ebert MS & Sharp PA 2012 Roles for microRNAs in conferring robustness to biological processes. Cell 149 515524. (https://doi.org/10.1016/j.cell.2012.04.005)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Evans JP 2020 Preventing polyspermy in mammalian eggs—contributions of the membrane block and other mechanisms. Molecular Reproduction and Development 87 341349. (https://doi.org/10.1002/mrd.23331)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fernandez-Gonzalez R, Laguna R, Ramos-Ibeas P, Pericuesta E, Alcalde-Lopez V, Perez-Cerezales S & Gutierrez-Adan A 2019 Successful ICSI in mice using caput epididymal spermatozoa. Frontiers in Cell and Developmental Biology 7 346. (https://doi.org/10.3389/fcell.2019.00346)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Flemr M, Malik R, Franke V, Nejepinska J, Sedlacek R, Vlahovicek K & Svoboda P 2013 A retrotransposon-driven dicer isoform directs endogenous small interfering RNA production in mouse oocytes. Cell 155 807816. (https://doi.org/10.1016/j.cell.2013.10.001)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gebert D, Neubert LK, Lloyd C, Gui J, Lehmann R & Teixeira FK 2021 Large Drosophila germline piRNA clusters are evolutionarily labile and dispensable for transposon regulation. Molecular Cell 81 39653978.e5. (https://doi.org/10.1016/j.molcel.2021.07.011)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Genzor P, Konstantinidou P, Stoyko D, Manzourolajdad A, Marlin Andrews C, Elchert AR, Stathopoulos C & Haase AD 2021 Cellular abundance shapes function in piRNA-guided genome defense. Genome Research 31 20582068. (https://doi.org/10.1101/gr.275478.121)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Girard A, Sachidanandam R, Hannon GJ & Carmell MA 2006 A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442 199202. (https://doi.org/10.1038/nature04917)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hasuwa H, Iwasaki YW, Au Yeung WK, Ishino K, Masuda H, Sasaki H & Siomi H 2021 Production of functional oocytes requires maternally expressed PIWI genes and piRNAs in golden hamsters. Nature Cell Biology 23 10021012. (https://doi.org/10.1038/s41556-021-00745-3)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Homolka D, Pandey RR, Goriaux C, Brasset E, Vaury C, Sachidanandam R, Fauvarque MO & Pillai RS 2015 PIWI slicing and RNA elements in precursors instruct directional primary piRNA biogenesis. Cell Reports 12 418428. (https://doi.org/10.1016/j.celrep.2015.06.030)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hutcheon K, McLaughlin EA, Stanger SJ, Bernstein IR, Dun MD, Eamens AL & Nixon B 2017 Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa. RNA Biology 14 17761790. (https://doi.org/10.1080/15476286.2017.1356569)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ishino K, Hasuwa H, Yoshimura J, Iwasaki YW, Nishihara H, Seki NM, Hirano T, Tsuchiya M, Ishizaki H & Masuda H et al.2021 Hamster PIWI proteins bind to piRNAs with stage-specific size variations during oocyte maturation. Nucleic Acids Research 49 27002720. (https://doi.org/10.1093/nar/gkab059)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Iwasaki YW, Siomi MC & Siomi H 2015 PIWI-interacting RNA: its biogenesis and functions. Annual Review of Biochemistry 84 405433. (https://doi.org/10.1146/annurev-biochem-060614-034258)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Johnson GD, Mackie P, Jodar M, Moskovtsev S & Krawetz SA 2015 Chromatin and extracellular vesicle associated sperm RNAs. Nucleic Acids Research 43 68476859. (https://doi.org/10.1093/nar/gkv591)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Jones HW Jr, Acosta AA, Andrews MC, Garcia JE, Jones GS, Mayer J, McDowell JS, Rosenwaks Z, Sandow BA & Veeck LL 1984 Three years of in vitro fertilization at Norfolk. Fertility and Sterility 42 826834. (https://doi.org/10.1016/s0015-0282(1648251-8)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Jung I, Park JC & Kim S 2014 piClust: a density based piRNA clustering algorithm. Computational Biology and Chemistry 50 6067. (https://doi.org/10.1016/j.compbiolchem.2014.01.008)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kohda T 2013 Effects of embryonic manipulation and epigenetics. Journal of Human Genetics 58 416420. (https://doi.org/10.1038/jhg.2013.61)

  • Kono T, Obata Y, Wu Q, Niwa K, Ono Y, Yamamoto Y, Park ES, Seo JS & Ogawa H 2004 Birth of parthenogenetic mice that can develop to adulthood. Nature 428 860864. (https://doi.org/10.1038/nature02402)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kuramochi-Miyagawa S, Watanabe T, Gotoh K, Totoki Y, Toyoda A, Ikawa M, Asada N, Kojima K, Yamaguchi Y & Ijiri TW et al.2008 DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. Genes and Development 22 908917. (https://doi.org/10.1101/gad.1640708)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lempradl A, Kugelberg U, Iconomou M, Beddows I, Nätt D, Casas E, Örkenby L, Enders L, Gutierrez Martinez A & Boonen E et al.2021 Intergenerational metabolic priming by sperm piRNAs. bioRxiv. (https://doi.org/10.1101/2021.03.29.436592)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Leung MR, Ravi RT, Gadella BM & Zeev-Ben-Mordehai T 2021 Membrane remodeling and matrix dispersal intermediates during mammalian acrosomal exocytosis. Frontiers in Cell and Developmental Biology 9 765673. (https://doi.org/10.3389/fcell.2021.765673)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li B, He X, Zhao Y, Bai D, Bou G, Zhang X, Su S, Dao L, Liu R & Wang Y et al.2019 Identification of piRNAs and piRNA clusters in the testes of the Mongolian Horse. Scientific Reports 9 5022. (https://doi.org/10.1038/s41598-019-41475-9)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li XZ, Roy CK, Dong X, Bolcun-Filas E, Wang J, Han BW, Xu J, Moore MJ, Schimenti JC & Weng Z et al.2013 An ancient transcription factor initiates the burst of piRNA production during early meiosis in mouse testes. Molecular Cell 50 6781. (https://doi.org/10.1016/j.molcel.2013.02.016)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li Y, Li RH, Ran MX, Zhang Y, Liang K, Ren YN, He WC, Zhang M, Zhou GB & Qazi IH et al.2018 High throughput small RNA and transcriptome sequencing reveal capacitation-related microRNAs and mRNA in boar sperm. BMC Genomics 19 736. (https://doi.org/10.1186/s12864-018-5132-9)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lim AK, Lorthongpanich C, Chew TG, Tan CW, Shue YT, Balu S, Gounko N, Kuramochi-Miyagawa S, Matzuk MM & Chuma S et al.2013 The nuage mediates retrotransposon silencing in mouse primordial ovarian follicles. Development 140 38193825. (https://doi.org/10.1242/dev.099184)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lopata A, Johnston IW, Hoult IJ & Speirs AI 1980 Pregnancy following intrauterine implantation of an embryo obtained by in vitro fertilization of a preovulatory egg. Fertility and Sterility 33 117120. (https://doi.org/10.1016/s0015-0282(1644529-2)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Loubalova Z, Fulka H, Horvat F, Pasulka J, Malik R, Hirose M, Ogura A & Svoboda P 2021 Formation of spermatogonia and fertile oocytes in golden hamsters requires piRNAs. Nature Cell Biology 23 9921001. (https://doi.org/10.1038/s41556-021-00746-2)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mao S, Goodrich RJ, Hauser R, Schrader SM, Chen Z & Krawetz SA 2013 Evaluation of the effectiveness of semen storage and sperm purification methods for spermatozoa transcript profiling. Systems Biology in Reproductive Medicine 59 287295. (https://doi.org/10.3109/19396368.2013.817626)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Nixon B, De Iuliis GN, Dun MD, Zhou W, Trigg NA & Eamens AL 2019 Profiling of epididymal small non-protein-coding RNAs. Andrology 7 669680. (https://doi.org/10.1111/andr.12640)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Nixon B, Stanger SJ, Mihalas BP, Reilly JN, Anderson AL, Tyagi S, Holt JE & McLaughlin EA 2015 The microRNA signature of mouse spermatozoa is substantially modified during epididymal maturation. Biology of Reproduction 93 91. (https://doi.org/10.1095/biolreprod.115.132209)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Oates AC 2011 What's all the noise about developmental stochasticity? Development 138 601607. (https://doi.org/10.1242/dev.059923)

  • Ohnishi Y, Totoki Y, Toyoda A, Watanabe T, Yamamoto Y, Tokunaga K, Sakaki Y, Sasaki H & Hohjoh H 2010 Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development. Nucleic Acids Research 38 51415151. (https://doi.org/10.1093/nar/gkq229)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ozata DM, Gainetdinov I, Zoch A, O’Carroll D & Zamore PD 2019 PIWI-interacting RNAs: small RNAs with big functions. Nature Reviews. Genetics 20 89108. (https://doi.org/10.1038/s41576-018-0073-3)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ozata DM, Yu T, Mou H, Gainetdinov I, Colpan C, Cecchini K, Kaymaz Y, Wu PH, Fan K