The agouti viable yellow (Avy) allele is an insertional mutation in the mouse genome caused by a variably methylated intracisternal A particle (VM-IAP) retrotransposon. This study casts light on the complex role of folate metabolism during development beyond a direct maternal effect. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. Moreover, the presence of conceptus skewing after embryo transfer into a control uterus indicates that misalignment is independent of the peri- and/or post-implantation uterus and instead is likely attributed to an embryonic mechanism that is epigenetically inherited. Genetic pedigree analysis reveals that severe conceptus skewing associates with the Mtrr genotype of either maternal grandparent. Multigenerational phenotype inheritance is characteristic of the Mtrr gt model, though the mechanism remains unclear. Since litters of Mtrr +/+ mothers display conceptus misalignment, a grandparental effect is explored. These observations argue against a post-implantation uterine defect as a cause of conceptus misalignment. While progesterone and/or BMP2 signalling might be disrupted, normal decidual morphology, patterning, and blood perfusion are evident at E6.5 regardless of conceptus orientation.
Misaligned conceptuses were observed in litters of Mtrr +/+, Mtrr +/gt, and Mtrr gt/gt mothers. To investigate a uterine effect of the Mtrr gt allele, we manipulate the maternal Mtrr genotype. Typically, the uterus is thought to guide conceptus orientation. Skewed growth likely influences embryo development since developmental delay and heart malformations (but not defects in neural tube closure or trophoblast differentiation) associate with severe misalignment of Mtrr gt/gt conceptuses.
Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. Additionally, some Mtrr gt/gt conceptuses display twinning.
The degree of misorientation occurs across a continuum, with the most severe form visible upon gross dissection. Notably, we report misalignment of some Mtrr gt conceptuses within their implantation sites from E6.5. We show that the spectrum of phenotypes previously observed in Mtrr gt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes. MTRR is necessary for methyl group utilisation from folate metabolism, and the Mtrr gt allele disrupts this process. Here, we explore how defective folate metabolism influences early development by analysing mice with the hypomorphic Mtrr gt mutation. Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear.