Poster Presentation ESA-SRB 2023 in conjunction with ENSA

The generation of a sperm-specific miR-30a/c2 overexpression mouse model for understanding paternal programming (#387)

Oluwabunmi I Olatunji 1 2 3 , Hannah E Lyons 1 3 , Victoria Nikitaras 1 3 , Mark Nottle 1 3 , Nicole McPherson 1 2 3
  1. University of Adelaide, Adelaide, SA, Australia
  2. Freemasons Centre for Male Health and Wellbeing, The University of Adelaide, Adelaide, South Australia, Australia
  3. Robinson Research Institute, The University of Adelaide, Adelaide, South Australia, Australia

Sperm specific microRNAs (miRs) delivered at fertilisation are causative mediators in paternal programming. MiR-30 is one of the most highly abundant miRs in sperm and expression levels are modified in circumstances of obesity, stress, heat, and infertility. MiR-30 have been shown to be a causative mediator in the transmission of paternal stress. However, current studies determining sperm miR-30 post fertilisation have occurred following synthetic microinjections, which lack important post transcription modifications acquired during testicular and epididymal transit that provide protection from scheduled RNA degradation post fertilisation. To overcome these limitations, we aimed to create a genetically modified mouse model that overexpressed miR-30a/c2 in sperm, without overtly disrupting spermatogenesis or sperm function.

Testes-specific CCNA1-EGFP-miR-30a/c2 construct were microinjected into zygotes and transferred into pseudo-pregnant females. Pups were screened by PCR for integration of CCNA1-EGFP construct to confirm inheritance and breeding maintained through the female line. Male offspring body composition (N=8) (total body weight, liver, kidneys pancreases, seminal vesicles etc.) sperm function (sperm count and motility via CASA) and testicular histology were assessed. The abundance of sperm miR-30a/c2 were assessed through TaqMan qPCR (N=4) following Trizol total RNA extraction from frozen sperm pellet.

Male mice carrying the transgene were of normal weight and had similar body composition to those of age-matched wildtype males (P>0.05). No changes were seen in sperm motility, sperm concentration, or testicular morphology between transgenic and wildtype mice (P>0.05). We observed a 10-fold increase in sperm miR-30a and miR-30c2 abundance in transgenic mice compared with wildtype (P<0.001 and P<0.01 respectively).  

 Over expression of miR-30a/c2 in the testes, resulted in increased expression in sperm without modifying reproductive traits or body composition. Our mouse model, overexpressing miR-30a/c2 in sperm can be utilized as a research model for recreating normal physiological responses seen in aberrant paternal programming.