Paternal factors significantly influence post-implantation embryo growth. For example, advanced paternal age at conception is linked to higher rates of fetal growth restriction, while babies born to obese males have increased body fat composition. However, the mechanisms by which paternal programming affects fetal growth remain unclear. Sperm-specific microRNAs (miRs) delivered during fertilisation have long been suspected to alter embryonic development, but their causal role in regulating fetal growth has yet to be established. Thus, we developed a novel mouse model of paternal programming that overexpresses sperm-specific miR-30a/c to demonstrate that these non-coding elements directly influence fetal phenotypes.
A testes-specific CCNA1-EGFP-miR-30a construct was microinjected into zygotes to create a mouse model with overexpression of sperm-specific miR-30a/c. To ensure our model did not produce off-target effects, we conducted a comprehensive assessment of whole-body composition, testes morphology, sperm motility kinetics (using a computer-assisted semen analyzer), and sperm quality metrics: reactive oxygen species (ROS) levels (MitoSOX Red) and mitochondrial membrane potential (JC-1) (n=4-8/generation). Males were then mated with 8-10-week-old females (N=10), with pregnancy rates and embryo development assessed at gestational age 18.5 days.
As anticipated, we observed a 4-fold increase in both miR-30a and 30c abundance in sperm of transgenic males (P<0.01), without modifying body composition, testicular morphology, or sperm characteristics (motility, morphology, ROS or mitochondrial membrane potential). Fetuses derived from sperm overexpressing miR-30a/c had reduced body weight (P=0.0003), resulting in reduced a fetal:placental weight ratio (P=0.03) compared with wildtype. Pregnancy rates, litter sizes, placental weight and diameter, and fetal crown-rump length were unchanged.
This is the first study to show a causative role of sperm miR30a/c in modulating in utero fetal growth, further providing evidence that sperm are contributing more than just DNA to the early embryo. Future work will focus on determining the contribution of sperm miR30a/c in offspring development.