A newly revealed “aging cliff” in sperm RNA marks a conserved molecular transition from youth to later life, offering fresh insight into how a father’s age may influence the earliest stages of development.
Study: Conserved shifts in sperm small non-coding RNA profiles during mouse and human aging. Image credit: WindNight/Shutterstock.com
A recent study in The EMBO Journal used a previously developed method, PANDORA-seq, to profile small non-coding RNAs (sncRNAs) in mouse and human spermatozoa across the lifespan, uncovering previously undetectable sncRNA species.
Impact of advanced paternal age on offspring
More men are becoming fathers later in life. Advanced paternal age reduces male fertility and increases health risks for children, including stillbirth, developmental disorders, and neuropsychiatric conditions. Animal studies have shown that offspring of older fathers are at higher risk of metabolic disorders, obesity, and anxiety.
Although the majority of studies on sperm aging have focused on DNA damage and methylation changes, scientists now recognize the epigenetic potential of small non-coding RNAs (sncRNAs) in sperm, which play a crucial role in the intergenerational transmission of paternal environmental cues and may influence early embryonic development.
The sncRNAs, including microRNAs (miRNAs), transfer RNA-derived small RNAs (tsRNAs), and ribosomal RNA-derived small RNAs (rsRNAs), carry information about a father’s age and experiences that may be transmitted to the next generation. They also serve as quality markers for embryos in fertility clinics, leading to the concept of a ‘sperm RNA code’, which refers to specific RNA patterns in sperm that are proposed to influence offspring health through paternal factors.
PANDORA-seq detects a hidden ‘aging cliff’ in mouse sperm
Researchers applied PANDORA-seq, a sequencing method that reduces detection bias caused by RNA modifications missed by traditional techniques, enabling analysis of tsRNAs and rsRNAs with chemical modifications. This technique revealed that miRNAs comprise less than 0.5 % of detected small RNA reads, while tsRNAs and rsRNAs are dominant and play key roles in paternal epigenetic transmission.
Analyzing mouse sperm across five age groups (10, 30, 50, 70, and 90 weeks), researchers found a distinct ‘aging cliff’ between 50 and 70 weeks, marked by a dramatic shift in tsRNA,rsRNA composition. This transition, which represents a population-level molecular shift rather than a single deterministic switch in individuals, separates early from late aging stages and was detectable only by PANDORA-seq. The tsRNA,rsRNA-based aging cliff showed stronger separation between early and late aging stages than the miRNA-based cliff, demonstrating PANDORA-seq’s superior sensitivity in detecting age-related changes.
Since sperm head RNAs are thought to be more functionally relevant for embryo development than cytoplasmic RNAs, researchers analyzed isolated sperm heads across the same age groups. Both whole sperm and sperm heads revealed an aging cliff at the 50–70 week transition using PANDORA-seq, confirming this as a robust aging signature.
Sperm heads contained mitochondrial tsRNAs and rsRNAs (0.14 % and 0.11 %) that showed coordinated age-related changes, mirroring the genomic aging cliff. This finding strongly suggests, but does not directly demonstrate, mitochondria-to-nucleus RNA transport as a potential mechanism mediating aging signals.
An age-related rsRNA length shift in sperm heads was noted, with longer RNAs increasing while shorter ones decreased, particularly in 28S- and 18S-derived rsRNAs. Mitochondrial rsRNAs showed similar trends, however tsRNAs did not. This length shift is consistent with a model in which aged sperm have reduced rsRNA processing capacity, potentially due to oxidative stress altering enzyme activity, with possible implications for fertility and offspring health.
Human sperm aging mirrors mouse rsRNA patterns
Researchers applied PANDORA-seq to human sperm from two cohorts: a longitudinal cohort of 8 donors sampled (ages 34–68), and a cross-sectional cohort of 47 donors (ages 25–51). Sperm heads were isolated to enrich aging signals and ensure RNA purity.
Both human cohorts revealed a consistent age-related shift in rsRNA length, mirroring mouse trends: longer rsRNAs increased, and shorter rsRNAs decreased. This shift was more prominent in total rsRNAs, specifically in 18S- and 28S-derived rsRNAs. Mitochondrial rsRNAs showed similar but weaker trends. These findings indicate that the rsRNA length shift is an evolutionarily conserved aging feature, which the authors propose may be linked to oxidative stress and altered enzymatic activity. Such changes may reshape the sperm RNA code and could impact embryo development and offspring health.
Assessing the functional significance of the sperm aging cliff
To test functional significance, researchers created RNA cocktails representing “young” and “old” sperm profiles using tsRNAs and rsRNAs with the most significant age-related expression changes. These cocktails were transfected into mouse embryonic stem cells, and gene expression was analyzed 24 hours later.
The old sperm RNA cocktail activated different genes than the young cocktail, particularly those involved in metabolism, mitochondrial function, and neurodegenerative diseases. These pathway changes align with biological processes implicated in health problems observed in offspring of older fathers, including metabolic and neurological disorders. This in vitro experiment demonstrates that age-related changes in sperm RNAs can directly alter gene activity in embryonic cells, providing proof-of-principle rather than direct evidence of in vivo inheritance or offspring phenotypes.
The authors also note that synthetic RNA cocktails may not fully recapitulate the biological activity of endogenous sperm RNAs, which naturally carry chemical modifications that can influence RNA stability and function.
Conserved sperm RNA aging marks a biological transition
Researchers used PANDORA-seq to discover an “aging cliff” in sperm, a sharp transition in small RNA patterns that occurs at a specific age in mice (50–70 weeks) and is reflected in conserved molecular features observed in human sperm. The current study found an age-related shift in rsRNA length: longer rsRNAs increase, while shorter ones decrease, with aging sperm.
This shift is proposed to reflect a reduced RNA processing capacity with age, potentially due to oxidative stress. Functional tests showed that RNA cocktails mimicking old sperm activated genes linked to metabolic disorders and neurological diseases in embryonic stem cells, supporting a model in which paternal age-associated changes in sperm RNA may influence early developmental gene regulation.
These conserved RNA signatures could serve as biomarkers for sperm quality in fertility clinics. They may, with further validation, inform future efforts to better understand paternal age-related reproductive and health risks.
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