Higgins, S. L. | bioRxiv Science

bioRxiv2026

Physiologic variation in sperm miRNAs tune embryonic gene regulatory programs and developmental outcomes

Small RNAs delivered by sperm can transmit environmentally regulated, epigenetically inherited phenotypes to offspring, yet the mechanisms by which modest changes in sperm microRNA abundance overcome dilution within the much larger egg to influence embryonic development remain unresolved. Here, we show that physiologically relevant variation in individual sperm miRNAs is sufficient to quantitatively program embryonic gene expression and developmental outcomes. Using parthenogenetic and fertilized embryos, we show that as few as 200 molecules of miR-200c-3p or miR-465c-3p induces reproducible, dose-dependent gene expression responses across defined developmental windows. Parthenogenetic embryos faithfully recapitulate early miRNA-driven gene expression changes observed in fertilized embryos, validating their use for isolating early regulatory mechanisms. We further developed AGO2-REMORA, an RNA adenosine base editor fused to Argonaute2 to map miRNA-mRNA interactions in embryos, revealing that early mRNA repression reflects direct miRNA targeting, while transcriptional changes at later stages arise as secondary consequences of these initial interactions. Furthermore, we show that modest elevation of miR-200c-3p during early development is sufficient to induce transcriptional alterations through early development and produce craniofacial phenotypes in late-stage embryos, recapitulating features of fetal alcohol syndrome associated with paternal alcohol consumption. Together, these findings establish a generalizable framework by which small perturbations in sperm miRNA content quantitatively modulate early gene regulatory programs, triggering cascades that persist throughout development and influence offspring phenotype.

developmental biology↗

bioRxiv2025

Therapy to Teratology: Chronic Paternal Antioxidant Supplementation Alters Offspring Placental Architecture and Craniofacial Morphogenesis in a Mouse Model

Oxidative stress plays a significant role in regulating the mammalian epigenome, with emerging evidence suggesting imbalances in the cellular redox state trigger stress-responsive epigenetic modifications that drive various human diseases. However, it remains unclear whether, like worms, epigenetic changes caused by redox imbalance or mitochondrial stress can move through the mammalian germline, potentially affecting the health of future generations. Antioxidant therapies are commonly used to reduce oxidative damage and are widely employed in cases of male infertility, where high-dose supplementation is often recommended to enhance sperm quality and overall measures of male reproductive health. Interestingly, in non-stressed, ostensibly healthy males, recent research suggests that antioxidants may have a negative influence on sperm epigenetic markers, indicating a potential epigenetic liability. However, whether male antioxidant treatment can induce paternal effects on offspring growth and development remains unknown. Here, we employed micro-CT imaging and geometric morphometrics to determine whether chronic antioxidant supplementation in healthy male mice affects placental growth and craniofacial development in their offspring. Adult C57BL/6J male mice were given a six-week preconception regimen of N-acetyl-L-cysteine (NAC; 400 mg/kg/day) and selenium (0.04 mg/kg/day), which continued throughout breeding with treatment-naive females. Although we observed modest alterations to the histological patterning of the female placenta, placental weights and efficiency remained unchanged. In contrast, we observed significant changes in facial shape and symmetry in both male and female offspring, with female offspring exhibiting significant reductions in eye spacing and head area. These changes occurred without any macro changes in paternal metabolic health, indicating that antioxidant-induced shifts in redox balance may disrupt developmental programming in the male germline independent of changes in overall health. Our findings emphasize the need for caution when using antioxidants as preconception interventions and broadly suggest that modulation of the paternal redox axis may result in altered developmental programming and teratogenic effects.

developmental biology↗