Sperm-specific microRNAs (miRs) are emerging mediators in paternal programming. Abundance levels of miR-30 are modified in circumstances of obesity, stress, heat, and infertility. But how it contributes to altered embryo development remains unknown. To investigate this, we generated a novel mouse model of sperm-specific miR-30a overexpression, and a zygote model of depleted miR30a.
Testes-specific CCNA1-EGFP-miR-30a construct were microinjected into zygotes to generate a sperm-specific miR30a overexpression mouse model. Zygotes were obtained by mating of superovulated CBAF1 females, with either wildtype (WT) or transgenic (TG) males. Alternatively, male CBAF1 mice were mated with super-ovulated CBAF1 females. At 16-18 h post-hCG injection, pronuclear staged embryos were collected and randomly allocated to either (i) culture only, (ii) microinjection of anti-miR-30a (0.05 ng/μL) into the male pronucleus (PN1-PN2), (iii) scrambled RNA control (0.05 ng/μL) or (iv) injection of vehicle controls (phosphate buffered saline (PBS). All embryos were cultured and assessed using established morphological criteria and blastocyst cell numbers assessed through antibody mediated differential staining.
We observed no differences in body composition or sperm parameters between WT and TG but observed an 8-fold increase in miR-30a abundance in TG sperm (P<0.01). Overexpression of miR-30a resulted in a reduced blastocyst rate, a delay in PN fading to 2-cell initiation and expanded blastocyst to hatching blastocyst (P=0.0346, 0.0189, 0.0467 respectively). Upon inhibiting miR-30a into the zygote, we see no differences in blastocyst development but observe a delay in time from pronuclear fading to 2-cell finish in anti-miR-30a group (P<0.05). Across both models, we observed decreased blastocyst total cell, inner cell mass and epiblast number (P<0.05) respectively from miR-30 modulation.
Dysregulation of miR-30a in the early embryo results in delayed blastocyst development and reduced cell numbers. Further studies will provide fundamental knowledge of paternal contribution to early embryo development.