Spermatocytogenesis, the mitotic phase of spermatogenesis, involves the differentiation of diploid spermatogonia into primary spermatocytes. Genomic stability is crucial during this stage, as errors in DNA repair or chromosome segregation can lead to infertility or defective offspring (Xie et al., 2021). The piRNA pathway plays an indispensable role in maintaining genomic integrity in mice, primarily by silencing TEs and regulating gene expression.

TEs constitute a substantial portion of the genome and, if left unchecked, can lead to genomic instability. Overactivation of TEs results in DNA damage, apoptosis, sperm abnormalities, and infertility. Specifically, pre-pachytene piRNAs, which are activated during embryogenesis and remain active postnatally, direct the de novo methylation of repetitive elements, ensuring epigenetic silencing. In mice, nuclear PIWI proteins such as MIWI2 mediate transcriptional silencing of TEs, while cytoplasmic PIWI proteins, including MILI and MIWI, regulate post-transcriptional silencing via the ping-pong cycle (Manakov et al., 2015).

Deficiencies in PIWI proteins often lead to early arrest during spermatogenesis. In Atlantic salmon, Almeida et al. reported that the loss of Piwil1 resulted in gametes deletion (Almeida et al., 2022). Some PIWI proteins also exhibit functional redundancy, in golden hamsters, PIWIL2 (cytoplasmic) can partially compensate for the absence of PIWIL4 in silencing specific TEs through the homotypic ping-pong pathway (Lv et al., 2023).

piRNA deficiencies also contribute to spermatogenic failure. In mice, Reddy et al. identified Pirmy and Pirmy-like RNAs as Y chromosome-derived noncoding transcripts that serve as templates for piRNAs, regulating genes critical for male fertility (Reddy et al., 2021). Notably, Y chromosome architecture and sequence content diverge markedly even among closely related species (e.g., Mus musculus vs. Mus caroli); thus, these findings may be context-specific to mice. Disruptions in these piRNAs, particularly due to Y chromosome deletions in the murine system, can cause abnormal sperm protein expression and male infertility (Reddy et al., 2021).

Deficiencies in the piRNA pathway exhibit species-specific phenotypic manifestations. Stallmeyer B et al. performed dual-species phenotypic analysis by characterizing individuals with biallelic high-impact variants in human piRNA pathway genes and generating corresponding knockout mouse models. Their investigation revealed fundamental interspecies divergence: defects in PIWIL2, PLD6, Glycerol-3-phosphate acyltransferase 2 (GPAT2), and TDRD12 produced profound phenotypic consequences in humans, whereas disruptions in TDRD9, Protein maelstrom homolog (MAEL), and PIWIL1 manifested more pronounced abnormalities in murine models. This differential phenotypic penetrance demonstrates that human and mice germlines possess distinct tolerance thresholds to impairments across the piRNA pathway machinery (Stallmeyer et al., 2024). “Trimming” is an important biochemical checkpoint in piRNA biogenesis, yet mutations in associated genes exhibit profound interspecies phenotypic divergence. Patients with PNLDC1 mutations uniformly demonstrate spermatogenic arrest at the pachytene spermatocyte stage, with only rare round spermatid production—representing significantly earlier developmental arrest than the elongated spermatid-stage arrest observed in Pnldc1 knockout mice. While murine models display severe molecular pathologies including profound reduction in MIWI-bound piRNAs and TE derepression, human PNLDC1 mutants exhibit globally extended piRNA lengths without systematically documented TE dysregulation or downregulation in PIWI-bound piRNAs, despite arresting at an earlier spermatogenic stage (Stallmeyer et al., 2024). This species-specific molecular manifestation needs further investigation into the different compensatory pathways in piRNA biogenesis between human and mice.

The piRNA pathway silences TEs through IMC and piP-body complexes (Li et al., 2021; Aravin et al., 2009). Primary piRNAs bind to MILI in prospermatogonia, cleaving TE transcripts to generate secondary piRNAs, which are subsequently loaded onto MILI and MIWI2, enabling nuclear translocation for transcriptional silencing (Aravin et al., 2009). Interspecies divergence characterizes the piRNA pathway’s TE silencing mechanisms. Mice prospermatogonia primarily employ piRNA-guided post-transcriptional degradation for TE suppression, with DNA methylation playing a comparatively limited role (Inoue et al., 2017). Conversely, during meiosis, DNA methylation emerges as the dominant silencing mechanism under the guidance of piRNA pathway proteins including SPOC domain-containing protein 1 (SPOCD1) and MIWI2(nuclear) (Inoue et al., 2017; Kawase and Ichiyanagi, 2022; Zoch et al., 2020). In Drosophila, the piRNA pathway operates through fundamentally distinct regulatory architecture: the Rhino-Deadlock-Cutoff (RDC) complex orchestrates transcription of dual-strand piRNA clusters, enforcing TE silencing at the transcriptional level via histone modification-mediated heterochromatinization (Chen et al., 2021c; Zhang et al., 2021). In humans, piRNAs focus on regulating primate-specific retrotransposable elements, like SINE-VNTR-Alus (SVAs), exhibiting more complex targeting mechanisms than in mice (Fukuda et al., 2022). This phenomenon may be related to humans having a higher proportion of transposons compared to mice (Goodier, 2016; Banuelos-Sanchez et al., 2019).

MOV10L1, a testis-specific RNA helicase, serves as a master regulator of piRNA biogenesis. Loss of MOV10L1 function results in pre-pachytene piRNA deficiency, retrotransposon activation, and ultimately, spermatogenic failure and infertility (Fu et al., 2016; Vourekas et al., 2015). Non-obstructive azoospermia is characterized by testicular failure despite patent genital ducts (Agarwal et al., 2021). In non-obstructive azoospermia patients, mutations in MOV10L1 have been linked to reduced piRNA levels and meiotic arrest (Wang et al., 2022b). MOV10L1 also interacts with ribosomes on primary piRNA transcripts, facilitating their translocation beyond stop codons, a process dependent on TDRD5 (Ding et al., 2018). A deficiency in MOV10L1 leads to primary transcripts accumulation and mature piRNA reduction, triggering retrotransposon activation and male infertility (Guan et al., 2021).

Additionally, MOV10L1 has a role in spermatogonia by silencing TEs through its RNA helicase activity and participates in miRNA-mediated regulation. In cytoplasmic RNA processing bodies (P bodies), MOV10L1 participates in post-transcriptional regulation (Zhu et al., 2015). A study by Fu et al. reported that MOV10 (MOV10L1’s paralog) deficiencies disrupted spermatogonial progenitor cells (SPCs), downregulating essential genes like ETS translocation variant 5 (Etv5), B-cell CLL/lymphoma 6 member B protein (Bcl6b), and Zinc finger and BTB domain containing 16 (Zbtb16), crucial for SPCs proliferation and self-renewal in mice germ cells (Fu et al., 2019). The disruption impairs SPCs’ ability to form seminiferous tubules, further emphasizing the importance of a fully functional piRNA pathway in spermatogenesis (Fu et al., 2019). Notably, in contrast to murine models, Mov10l1 deficiency in golden hamsters results in infertility in both sexes, indicating a critical species-specific divergence in piRNA pathway functionality within female reproductive biology (Hasuwa et al., 2021; Loubalova et al., 2021).

Other proteins involved in the piRNA pathway also play crucial roles in genomic integrity. For instance, in mice, Gametocyte-specific factor 1 (GTSF1) and TDRD9 coordinate transcriptional silencing, with GTSF1 loss leading to depression of long interspersed element 1 (LINE-1)and intracisternal A particle (IAP) retrotransposons in prospermatogonia (Yoshimura et al., 2018). Testis-expressed protein 15 (TEX15), a testis-specific nuclear protein essential for the DNA damage response, works alongside MILI(nuclear) to recruit epigenetic silencing machinery to transposon loci (Yang et al., 2020). Notably, TEX15 is vital for spermatogenesis, but its deficiency does not disrupt piRNA biogenesis (Schopp et al., 2020). Finally, Adenosine deaminase domain-containing protein 2 (ADAD2), a protein highly expressed in male germ cells, regulates piRNA populations in mice. ADAD2 is a key player in the piRNA biogenesis interacting with multiple RNA-binding proteins, including MILI, MIWI, and RING finger protein 17 (RNF17). RNF17 preferentially binds to long precursors of pachytene piRNA clusters, and competitively reducing the opportunity for transposon RNA to interact with PIWI proteins, thereby preventing the over-activation of the ping-pong activity (Wasik et al., 2015). Adad2 deficiency markedly reduces the formation of RNF17 granules, causing the aberrant activation of the piRNA ping-pong cycle, resulting in a higher proportion of secondary piRNAs with a 10th A bias. Deficiency in Adad2 also results in elevated expression of piRNAs derived from transposons, while the cluster-derived pachytene piRNAs significantly decrease, including those associated with MILI and MIWI. This alteration closely resembles the abnormal piRNA patterns observed with RNF17 loss, suggesting a synergistic role of ADAD2 and RNF17 in regulating the genomic source preference of piRNAs (Lu et al., 2023).

Beyond maintaining genomic integrity, piRNAs also respond to testicular damage from environmental stressors. Chen et al. showed that heat stress in mice upregulates 88 piRNA clusters and downregulates 47 piRNA clusters (Chen et al., 2023). Key piRNA piR-020492 is significantly upregulated, affecting AMP-activated protein kinase (AMPK) and insulin pathways, inhibiting germ cell proliferation, and inducing apoptosis (Chen et al., 2023). Similarly, Zhang et al. found that exposure to Microcystin-LR (MC-LR) elevates piR_003399, causing cytotoxicity in spermatogonia in mice (Zhang et al., 2018). Suppressing piR_003399 increased Cyclin-dependent kinase 6 (CDK6) levels, improving sperm motility and cell cycle progression in mice (Zhang et al., 2018). piR_003399 levels in serum and plasma exhibited dose-correlated variations with MC-LR exposure, paralleling the severity of reproductive impairment in mice in vivo models (Zhang et al., 2018). This supports its utility as a potential circulating biomarker for MC-LR-induced reproductive toxicity. These findings highlight piRNAs’ critical role in protecting testicular health.

In conclusion, the piRNA pathway is indispensable for maintaining genomic stability during spermatocytogenesis by silencing TEs and regulating essential gene expression. Disruptions in this pathway can lead to severe spermatogenic failure and infertility.

Spermatidogenesis, the second phase of spermatogenesis, involves the transformation of spermatocytes into spermatids through two meiotic divisions. This process begins with Meiosis I, where primary spermatocytes undergo chromosomal recombination and pairing during prophase, then divide into two secondary spermatocytes; Meiosis II follows, producing four haploid spermatids from each primary spermatocyte (Ishiguro, 2024). These spermatids are immature, round or oval-shaped cells lacking motility. The role of piRNAs in this phase is critical for maintaining genomic integrity and ensuring successful meiosis (Newkirk et al., 2017).

PIWI proteins play a central role in RNA cleavage, essential for reproductive function during meiosis. De Fazio et al. found that the RNA-cleaving activity of MILI relies on the conserved disulfide-directed β-hairpin fold (DDH) (Asp-Asp-His) motif in mice (De Fazio et al., 2011). Mutations in this motif (e.g., DAH or ADH) weakened MILI(cytoplasmic)’s cleavage activity, disrupting piRNA production, failing to silence TEs, and leading to spermatogenic arrest during meiosis. Co-factors, such as GTSF1, enhance MIWI(cytoplasmic)’s RNA cleavage function, while DEAD box polypeptide 4 (DDX4) and DDX43 promote the release of cleavage products, accelerating RNA degradation (Arif et al., 2022). Hsieh et al. further demonstrated that piRNAs, such as piR major-a and piR major-b, guide MIWI to cleavage sites, ensuring correct kinetochore assembly and chromosomal separation during meiosis in mice (Hsieh et al., 2020). Similarly, Chen et al. identified sex-specific piRNAs in Drosophila melanogaster that guide transcript cleavage, achieving gene regulation in the testis (Chen et al., 2021b).

Pachytene piRNAs play a central role in RNA cleavage. Cecchini K et al. demonstrated that the core function of murine pachytene piRNAs is to regulate target mRNAs through endonucleolytic cleavage, distinct from miRNA-like mechanisms or transcriptional silencing (Cecchini et al., 2024). Although the majority of these cleavage events do not alter the steady-state abundance of their target mRNAs, the regulation of a limited subset of critical mRNAs is indispensable for male fertility. Furthermore, transposon-derived piRNAs account for a higher proportion of targeting events, yet the high transcriptional activity of most targets buffers the impact of cleavage on their overall abundance. The evolutionary conservation of pachytene piRNAs is likely driven by selective advantages conferred by a minority of functional piRNAs, rather than the activity of the entire population—a feature potentially intrinsic to pachytene piRNA biology.

The piRNA pathway enforces TE silencing via species-specific epigenetic mechanisms—including DNA methylation in mice and histone modifications in Drosophila—with disruptions causing germ cell arrest and male infertility. PIWI proteins collaborate with SPOCD1 to establish methylation-dependent silencing of retrotransposons (Zoch et al., 2020). In the murine system, SPOCD1 interacts with MIWI2 and recruits the DNA (cytosine-5)-methyltransferase 3-like protein (DNMT3L)-DNA (cytosine-5)-methyltransferase 3A protein (DNMT3A) methyltransferase complex to nascent transposon loci, facilitating repressive chromatin remodeling. While de novo DNA methylation is largely restricted to embryonic germ cell development, this machinery may primarily enforce maintenance methylation during spermatidogenesis in mice, ensuring persistent TE suppression. Zoch et al. demonstrated that SPOCD1 deficiency disrupts LINE1 and IAP silencing, leading to meiotic arrest and male infertility, despite intact piRNA biogenesis and MIWI2(nuclear) localization (Zoch et al., 2020).

Nuclear protein E3 ubiquitin-protein ligase UHRF1, a five-domain epigenetic regulatory factor, plays a pivotal role in coordinating the piRNA pathway with chromatin remodeling machinery (Dong et al., 2019). UHRF1 interacts with arginine methyltransferase Protein arginine N-methyltransferase 5 (PRMT5), which regulates histone arginine modifications (symmetric dimethylation of histone H4 on Arg 3 (H4R3me2s) and symmetric dimethylation of histone H3 at arginine 2 (H3R2me2s)) (Wang et al., 2015). UHRF1 also controls the localization of key PIWI pathway proteins (MILI, MIWI, and TDRKH), thereby regulating piRNA biogenesis in mice (Li et al., 2021). Dong et al. discovered that UHRF1 forms a complex with PRMT5 and MILI/MIWI in the cytoplasm of mouse spermatocytes, enhancing the cleaving activity of PIWI proteins, which contributes to the post-transcriptional suppression of retrotransposons (Dong et al., 2019). In Uhrf1-deficient mice, piRNA pathway dysfunction results in the loss of pachytene piRNAs, impaired cooperation with PRMT5, global DNA demethylation, retrotransposon upregulation, activation of the DNA damage response, and altered chromatin states, ultimately leading to male infertility (Dong et al., 2019).

The piRNA pathway exhibits interspecies divergence in its mechanisms for TE suppression. FKBP Prolyl Isomerase Family Member 6 (Fkbp6) has been implicated in secondary piRNA biogenesis and synaptonemal complex assembly in mice, with its deficiency leading to meiotic arrest (Xiol et al., 2012; Crackower et al., 2003). In murine models, Fkbp6 loss disrupts synaptonemal complex formation and activates LINE-1 retrotransposons (Wyrwoll et al., 2022). In contrast, human cases of FKBP6 deficiency also cause spermatogenic failure but lack the pronounced LINE-1 derepression observed in mice, suggesting that humans may employ more complex redundant mechanisms and fine-tuning capabilities for TE silencing within the piRNA pathway (Wyrwoll et al., 2022). This phenotypic divergence is similarly observed in TDRD5 deficiency. While loss of Tdrd5 in mice causes retrotransposon derepression and meiotic arrest (Yabuta et al., 2011), human testes harboring TDRD5 mutations exhibit normal LINE-1 open reading frame 1 protein (LINE-1-ORF1p) expression with no observation of TE activation (Guo et al., 2025).

Notably, DNA methylation is not a universal TE silencing mechanism across species. For instance, in Drosophila, DNA methylation associated with the PIWI/piRNA pathway has not been reported. Instead, piRNA-mediated silencing relies predominantly on histone modifications (e.g., trimethylation of histone H3 lysine 9 (H3K9me3)) rather than DNA methylation (Zhang et al., 2021). Thus, the SPOCD1-MIWI2-DNMT3 axis described here reflects a murine-specific adaptation (Zoch et al., 2020). The uncharacterized protein C19ORF84 further bridges SPOCD1 with the methylation machinery, reinforcing piRNA-guided epigenetic silencing in mice. These findings underscore the importance of species context when interpreting piRNA-mediated TE control mechanisms (Zoch et al., 2024).

TE silencing mediated by piRNAs may also be achieved via chromatin modification pathways during spermatidogenesis. Research by Mahadevan et al. highlighted the role of histone H1t in chromatin regions containing TEs, where it interacts with repressive epigenetic markers and proteins, suggesting a mechanism for piRNA-mediated recruitment of repressive chromatin factors in mouse pachytene spermatocytes (Mahadevan et al., 2020). Under the regulation of piRNAs, H1t might facilitate the suppression of TEs through the induction of localized chromatin relaxation, which enables the recruitment of heterochromatin-associated proteins and repeat repressive-associated protein factors, ultimately leading to the formation of closed chromatin repressive structures (Mahadevan et al., 2020). Additionally, piRNA clusters are enriched with the histone variant H3.3B, which is crucial for spermatogenesis. Loss of H3f3b in mice severely impairs piRNA cluster transcription during meiosis, leading to infertility due to defective post-meiotic cells (Fontaine et al., 2022).

During spermatidogenesis, piRNAs also guide mRNA translation. Dai et al. identified specific MIWI-CLIP clusters on piRNA target sites in key genes like Plectin, Arf-GAP domain and FG repeat-containing protein 1 (Agfg1), TATA box-binding protein-like 1 (Tbpl1), CCR4-NOT transcription complex subunit 4 (Cnot4), and Ubiquitin-like protein ATG12 (Atg12), which are crucial for acrosome formation and spermatid development in mice (Dai et al., 2019). These mRNAs contain AU-rich elements (AREs) in their 3′UTRs, necessary for piRNA-mediated translational activation. This process also inhibits the repressive actions of specific piRNAs, like piR_010111, on some genes. piRNA-mRNA interactions guide MIWI binding to these loci, assembling the MIWI/eIF3f/HuR complex to promote translation. Wang et al. further demonstrated that extended piRNA-mRNA complementarity in mice enhances RNA-binding protein human antigen R (HuR) binding to target mRNA in an ARE-independent manner, expanding the regulatory scope of the piRNA pathway (Wang et al., 2023b).

The interaction between piRNAs and the ubiquitin-proteasome system (UPS) represents an emerging area of research, particularly in the contexts of germline development and cancer. The piRNA pathway has been linked to post-translational protein degradation and apoptosis—the ubiquitin (Ub) pathway. Ub is a highly conserved protein crucial for fertility in both male and female mice (Martin-Villanueva et al., 2021). In mice, the polyubiquitin gene Polyubiquitin-B (Ubb) is composed of a tandem repeat of four ubiquitin-coding units; its primary function is to maintain cellular ubiquitin homeostasis by encoding tandemly repeated ubiquitin units and regulate critical physiological processes (Martin-Villanueva et al., 2021). Further mechanistic studies demonstrated that Ubb deficiency disrupts ubiquitin homeostasis, triggering a cascade of pathological events: (i) depletion of the free ubiquitin pool impairs proteasomal degradation, resulting in abnormal protein accumulation; (ii) dysregulation of germline-specific genes (Ddx4, Tdrd6, Tdrd7, Rnf17) and piRNA pathway effectors (MIWI, MILI) compromises post-transcriptional regulation; and (iii) synergistic dysfunctions culminate in pachytene-stage meiotic arrest, germ cell apoptosis, and complete gametogenesis failure (Han et al., 2021). Charmant O et al. revealed that the ubiquitination of the Paramecium PIWI protein is indispensable in male fertility. Ptiwi09 is predominantly regulated by Gtsf1 (Charmant et al., 2025). This process is initiated when Gtsf1 interacts with the Ptiwi09-maternal polyploid somatic macronucleus (MAC)-scnRNA complex, which is paired with nascent non-coding transcripts from the MAC. This binding triggers Ptiwi09 ubiquitination, facilitating the degradation of both the Ptiwi09 protein and its associated MAC-scnRNAs, compromising genome integrity. In Gtsf1-KD Paramecium, a marked reduction in Ptiwi09 ubiquitination is observed, resulting in abnormal MAC-scnRNA levels and TE activation (Charmant et al., 2025).

Small ubiquitin-like modifier (SUMO) is a highly conserved post-translational modifier that expands the functional diversity of the eukaryotic proteome through dynamic conjugation to target proteins (Celen and Sahin, 2020). Recent studies have revealed an intimate mechanistic link between SUMOylation and the piRNA pathway in female reproductive system. In D. melanogaster, diGly-based proteomic profiling has uncovered widespread SUMOylation of core piRNA pathway components, including nuclear factors such as Piwi and Panoramix (Panx), as well as cytoplasmic nuage constituents like Spindle-E (Spn-E) and Mael (Ninova et al., 2023). Notably, Piwi differentially regulates the SUMOylation status of these substrates, suggesting a hierarchical SUMO-dependent architecture within the piRNA pathway (Ninova et al., 2023). Functional ablation of SUMO disrupts the integrity of nuage, transforming perinuclear Vasa- and Aub-positive granules into dispersed puncta, thereby impairing piRNA biogenesis and post-transcriptional silencing of TEs (Ninova et al., 2023). Parallel work done in hermaphrodite C. elegans has demonstrated that SUMOylation of the germline determinant pharynx and intestine in excess protein 1 (PIE-1) is also essential for both germline fate maintenance and piRNA-mediated TE repression, underscoring the evolutionary conservation of this regulatory axis (Kim et al., 2021).

Mechanistically, SUMOylation acts as a molecular switch linking piRNA-guided silencing to chromatin remodeling. Ninova M et al. discovered that in Drosophila, the SUMO E3 ligase Su(var)2-10 interacts directly with the Piwi–Panx–Arx complex and facilitates the SUMOylation of both itself and associated chromatin factors (Ninova et al., 2020). This modification promotes recruitment of the SetDB1/Windei histone methyltransferase complex, enabling deposition of H3K9me3 marks at transposon loci and transcriptional repression of approximately 60% of TE families (Ninova et al., 2020). These findings establish SUMOylation as an essential scaffold for chromatin-based transposon control, effectively coupling small RNA recognition to epigenetic enforcement.

While the role of SUMOylation in piRNA-mediated silencing is well established in model organisms, its relevance in mammals and its interplay with other post-translational modifications are less clear. Although the role of SUMO in the piRNA pathway has been well studied in the female reproductive system, its function in male germ cells requires further investigation. Additionally, the dynamic regulation of SUMOylation and deSUMOylation, and their impact on piRNA pathway and condensate formation, are in need of further exploration.Taken together, the piRNA pathway is crucial for spermatidogenesis, maintaining genomic integrity, controlling protein degradation, regulating mRNA translation and apoptosis—all essential for the proper development and maturation of spermatids.

Spermiogenesis, the final stage of spermatogenesis, exhibits marked species-specificity in its morphological and molecular progression. This stage contains key molecular events including: proacrosomal vesicle formation, histone-to-protamine transition, acrosome biogenesis flagellar assembly, spermiation and epididymal maturation (Hess and Renato de Franca, 2008). The piRNA pathway and its functional partners regulate critical aspects of murine spermiogenesis, including transposon silencing during nuclear condensation and mRNA surveillance in cytoplasmic droplets.

The primary biological function of piRNAs in spermiogenesis is TEs silencing, particularly LINE1 (L1) retrotransposons, which make up about 20% of the mammalian genome (Doucet et al., 2015). L1 retrotransposition involves RNA polymerase II (RNAPII) synthesizing L1 RNA, which is then cleaved and polyadenylated. The transcript is exported to the cytoplasm and translated into two proteins: ORF1 and ORF2. ORF1 packages L1 RNA for nuclear import, while ORF2 facilitates reverse transcription and integration of L1 into the genome (Miyoshi et al., 2019). piRNA-mediated silencing of L1 is crucial for preventing spermatogenic arrest and infertility in male germ cells (Yang and Wang, 2016). In Pnldc1-deficient mice, piRNA trimming is disrupted, leading to L1 depression and spermatogenic arrest (Ding et al., 2017). TDRKH and PNLDC1 modulate MIWI localization in chromatoid bodies (CBs), promoting L1 silencing in round spermatids (Wei C. et al., 2024). Mutations in the Rhox gene cluster, encoding transcription factors critical for reproductive health, have also been linked to L1 silencing (Tan et al., 2021) (Table 1).

In C. elegans, the cytoplamsic Argonaute protein CSR-1 antagonizes piRNA-mediated silencing by promoting the expression of neo-formed RNAs, protecting germline-expressed genes (Cornes et al., 2022). This regulation facilitates the precise timing of piRNA silencing functions during spermiogenesis. piRNA biogenesis and degradation are tightly controlled, as seen in silkworms, where small RNA 2′-O-methyltransferase (BmHen1) and BmPnldc1 are essential for piRNA stability and TE silencing (Yang et al., 2022).

Chromatin remodeling, particularly the histone-to-protamine transition, is another critical process regulated by the piRNA pathway. PIWI proteins, in cooperation with piRNAs, regulate histone modifications during spermiogenesis. In Drosophila, CDS-piRNAs regulate genes involved in histone acetylation, such as newly excysted juveniles (nej), affecting histone acetyltransferase activity (Iki et al., 2023). Loss of aubergine(aub), a PIWI-interacting protein, leads to excessive histone acetylation and disrupted histone-protamine transitions, resulting in defective spermiogenesis (Iki et al., 2023). The THO RNA export complex and Gtsf1 are also involved in piRNA-guided chromatin modifications, such as H3K9me3, to ensure TE silencing during spermatogenesis in Drosophila (Donertas et al., 2013).

The elimination of PIWI proteins is closely linked to histone ubiquitination. Specifically, MIWI and RNF8 regulate H2B ubiquitination and MIWI degradation, both of which are essential for completing spermatogenesis in mice (Gou et al., 2017). MIWI acts as a molecular switch, initiating the histone-to-protamine transition critical for sperm maturation. Disruption of MIWI or RNF8 expression leads to accumulation of MIWI in cytoplasm and male infertility due to abnormal sperm morphology. piRNAs mediate PIWI clearance to ensure successful spermatogenesis (Gou et al., 2017). Zhao et al. found that in late-stage mouse spermatids, MIWI is degraded via the anaphase promoting complex/cyclosome (APC/C)-26S proteasome pathway, and piRNAs facilitate the interaction of MIWI with APC/C substrate subunits. This piRNA-triggered de-ubiquitination and MIWI degradation promote piRNA clearance, aiding sperm maturation (Zhao et al., 2013).

Nuclear condensation and cytoplasmic exclusion represent critical terminal events in spermiogenesis, essential for functional sperm maturation. Nuclear compaction achieves dramatic volumetric reduction of the spermatid nucleus through hyper-condensation of chromatin, forming the streamlined sperm head architecture. Concurrently, cytoplasmic exclusion eliminates superfluous organelles and transcripts via sequestration into residual bodies, purging most of cytoplasmic volume (Hess and Renato de Franca, 2008). This concerted remodeling enhances sperm motility while eliminating redundant mRNAs/proteins that could compromise early embryogenesis. Pachytene piRNAs provide the molecular logic underpinning these transformations. Through pi-RISC mediated large-scale mRNA clearance, they terminate dispensable cellular programs, enabling structural reorganization. Gou LT et al. demonstrated that murine pachytene piRNAs form chromatin assembly factor 1 (CAF1)-containing pi-RISCs in elongating spermatids. These complexes employ imperfect base-pairing to bind 3′UTRs of target mRNAs (e.g., G protein-coupled receptor kinase 4 (Grk4), Transcription factor SOX-6 (Sox6)), where CAF1 catalyzes deadenylation-independent decay. Crucially, this degradation operates independently of MIWI’s slicer activity, but requires MIWI’s piRNA-loading competence (Gou et al., 2014). By orchestrating transcriptome-wide silencing, pachytene piRNAs facilitate nuclear compaction and cytoplasmic exclusion–establishing the molecular framework for spermatid-to-spermatozoon transformation.

The piRNA pathway is also responsive to environmental factors that affect chromatin remodeling. Liu et al. demonstrated that exposure to silicon dioxide nanoparticles (SiNPs) alters MIWI expression patterns in mice spermatocytes, particularly in round spermatids (Liu et al., 2021). This increased MIWI expression can cause RNF8 retention in the sperm cell cytoplasm, leading to downregulated histone (H2A/H2B) removal and inhibited formation of ubH2A and ubH2B (Liu et al., 2021). The disrupted exchange of histone-to-protamine and chromatin condensation ultimately affecting the round spermatid differentiation and leading to sperm abnormalities (Liu et al., 2021). The study offers insights into how environmental contaminants like SiNPs negatively impact reproductive health. The piRNA pathway is essential for acquiring sperm motility, and deficiencies in piRNAs are often associated with impaired sperm vitality and morphology. Choi et al. found that mice lacking the pachytene piRNA cluster on chromosome 18 (pi18) exhibited severe sperm abnormalities in mice, including malformed heads, excessive acrosome formation, impaired acrosome exocytosis, defects in the axonemal complex of the mitochondrial sheath, and partial loss of outer dense fibers (ODFs) in the tail (Choi et al., 2021). These abnormalities led to poor sperm motility, reduced activity, and infertility. The absence of pi18 primarily disrupted the structural integrity of the trans-Golgi network, which was associated with enlarged proacrosomal vesicles and upregulation of Golgin subfamily A member 2 (GOLGA2) transcripts and protein (Choi et al., 2021).

Furthermore, Kong et al. discovered that piR_32362259 may influence the development of sperm damage by regulating the phosphatidylinositol 3-kinase (PI3K) - protein kinase B (AKT) signaling pathway in mice (Kong et al., 2021). Lower expression levels of this piRNA were found to slow the reduction in sperm vitality, influence the cell cycle, and decrease apoptosis rates. In mice, Wu et al. identified that mutations in pi6 piRNAs led to defective Ca²⁺ influx during the acrosome reaction, impairing the sperm’s ability to penetrate or bind to the zona pellucida, a critical step in fertilization (Wu et al., 2020).

Pachytene piRNAs are critically involved in maintaining sperm motility. The clusters pi9 and pi17 serve as major sources of murine pachytene piRNAs, collectively accounting for 13.5% of the total pool. Their loss ablates the production of corresponding pachytene piRNAs and damages RNA cleavage activity (Gainetdinov et al., 2023). Cecchini K et al. generated pi9 ^−/− and pi17 ^−/− single mutant male mice, which exhibited only mild reductions in sperm motility and remained fertile—indicating genetic redundancy between these loci. In contrast, pi9 ^−/− pi17 ^−/− double mutants displayed severe phenotypes, exhibiting significantly reduced sperm count and motility, loss of zona pellucida penetration ability, and complete infertility (Cecchini et al., 2024). This study underscores the critical role of pachytene piRNAs in regulating sperm function.

Additionally, Cornes et al. demonstrated that C. elegans spermatocytes lacking PIWI or Argonaute protein HRDE-1 failed to produce pseudopod-like structures observed in wild-type spermatocytes, leading to reduced sperm motility and significantly fewer offspring when crossed with wild-type individuals (Cornes et al., 2022). This highlights the critical role of piRNAs in maintaining sperm motility and ensuring reproductive success. Taken together, specific piRNAs and PIWI/piRNA pathway-associated proteins are involved in different stages of spermatogenesis (Figure 3).

The Ago family proteins serve as central executors across all three pathways. Ago proteins universally bind small RNAs to form the RNA-induced silencing complex (RISC), mediating gene silencing through translational repression and mRNA decay (Azlan et al., 2016). Certain Ago proteins (e.g., human AGO1/2, Drosophila DmAGO2) localize to the nucleus, where they exert additional regulatory functions by promoting DNA methylation and modulating alternative splicing (Tan et al., 2009; Cernilogar et al., 2011; Azlan et al., 2016).

The methyltransferase HEN1 plays a conserved yet pathway-specific role in small RNA stabilization across species. Horwich MD et al. demonstrated that in Drosophila, the methyltransferase activity of DmHen1 majorly functions in the piRNA and siRNA pathways (Horwich et al., 2007). DmHen1 critically maintains piRNA stability and function by catalyzing 3′-terminal 2′-O-methylation in germ cells (Horwich et al., 2007). Within the siRNA pathway, DmHen1 similarly methylates the 3′end of single stranded siRNAs following their loading into Ago2-RISC (Horwich et al., 2007). Intriguingly, a subset of Ago2-bound miRNAs (e.g., miR-277) also undergo DmHen1-mediated methylation (Horwich et al., 2007). This functional conservation extends to the C. elegans HEN1 ortholog henn-1 (Montgomery et al., 2012). Conversely, in N. vectensis, Hen1 primarily methylates miRNAs and piRNAs (Modepalli et al., 2018). Notably, knockdown of PIWI2 in Nematostella vectensis not only reduced piRNA levels but unexpectedly caused a significant decrease in miRNA abundance, suggesting piRNA pathway involvement in miRNA homeostasis, potentially via broad transcriptional regulation (Modepalli et al., 2018).

Emerging evidence indicates bidirectional crosstalk between these pathways. In Drosophila, Iki T et al. demonstrated that siRNAs can directly initiate piRNA biogenesis: 3′fragments generated by siRNA-directed cleavage of ATPsynbeta mRNA are loaded onto Aub and processed into CDS-piRNAs, evidenced by 5′-end complementarity between the siRNAs and resultant piRNAs (Iki et al., 2023). Furthermore, miRNAs (e.g., miR-316) bound to Ago2 can specify piRNA precursor regions by recognizing antisense sequences within target transcripts (e.g., muc14A), positioning Ago2 to initiate CDS-piRNA biogenesis (Iki et al., 2023).

miRNAs and piRNAs engage in reciprocal regulatory loops critical for developmental processes. Du WW et al. revealed that during murine embryonic development, miR-17-5p disrupts TE silencing by inhibiting the piRNA ping-pong amplification cycle and downregulating Mili and Dnmt3a. Conversely, injection of piR-11 into zygotes competitively attenuated miR-17-5p-mediated suppression of Mili and Dnmt3a, demonstrating mutual regulation essential for normal embryogenesis and TE control (Du et al., 2016).

piRNA-siRNA cooperation is evolutionarily conserved for robust gene silencing. High-throughput sequencing in four nematodes (C. elegans, Caenorhabditis briggsae, Caenorhabditis remanei, Caenorhabditis brenneri) revealed a conserved mechanism where piRNAs guide AGO proteins to cleave target mRNAs, triggering RdRP-dependent amplification of secondary siRNAs (Shi et al., 2013). In C. elegans, Manage KI et al. elucidated that piRNAs bound to the PIWI homolog PRG-1 recognize target mRNAs within P granules (Manage et al., 2020). This initial recognition, facilitated by the Tudor domain protein SIMR-1, nucleates secondary siRNA biogenesis, establishing a synergistic “piRNA-priming, siRNA-amplification” cascade essential for silencing target mRNAs, maintaining genomic integrity, and ensuring proper germ cell development (Manage et al., 2020).

piRNAs, miRNAs, and siRNAs form an interconnected regulatory network through shared biogenesis factors, reciprocal modulation, and overlapping effector mechanisms. These intricate interactions are crucial for fine-tuning gene expression, particularly during germline development and early embryogenesis. Understanding these relationships is fundamental to elucidating how small RNA pathways coordinate to maintain genomic integrity and regulate complex developmental programs.