Disruption of Folate Metabolism Causes Poor Alignment and Spacing of Mouse Conceptuses for Multiple Generations

Abstract

Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear. Here, we explore how defective folate metabolism influences early development by analysing mice with the hypomorphic Mtrrgt mutation. MTRR is necessary for methyl group utilisation from folate metabolism, and the Mtrrgt allele disrupts this process. We show that the spectrum of phenotypes previously observed in Mtrrgt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes. Notably, we report misalignment of some Mtrrgt conceptuses within their implantation sites from E6.5. The degree of misorientation occurs across a continuum, with the most severe form visible upon gross dissection. Additionally, some Mtrrgt/gt conceptuses display twinning. Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. 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 Mtrrgt/gt conceptuses. Typically, the uterus is thought to guide conceptus orientation. To investigate a uterine effect of the Mtrrgt allele, we manipulate the maternal Mtrr genotype. Misaligned conceptuses were observed in litters of Mtrr+/+, Mtrr+/gt, and Mtrrgt/gt mothers. 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. These observations argue against a post-implantation uterine defect as a cause of conceptus misalignment. Since litters of Mtrr+/+ mothers display conceptus misalignment, a grandparental effect is explored. Multigenerational phenotype inheritance is characteristic of the Mtrrgt model, though the mechanism remains unclear. Genetic pedigree analysis reveals that severe conceptus skewing associates with the Mtrr genotype of either maternal grandparent. 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. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. This study casts light on the complex role of folate metabolism during development beyond a direct maternal effect.

One-carbon metabolism is required for epigenetic stability in the mouse placenta

“…We carried out high-throughput sequencing of immunoprecipitated methylated DNA (meDIP-seq) from C57Bl/6J control and Mtrr gt/gt placentas at E10.5. Mtrr gt/gt placentas were divided into two phenotypic groups including those from fetuses that were phenotypically normal (PN) or were FGR based on crownrump length [10,22]. Placentas from C57Bl/6J mice were controls since the Mtrr gt allele was backcrossed into the C57Bl/6J genetic background [10].…”

“…Conceptuses were rigorously scored for gross phenotypes during dissection and allocated to the phenotypic categories that were previously defined, including phenotypically normal (PN), fetal growth enhancement (FGE), fetal growth restriction (FGR), developmental delay, severe abnormalities (e.g., congenital heart defects, neural tube closure defects, hemorrhages, skewed conceptus orientation, twinning, etc), and resorption [10,22]. Notably, conceptuses with >1 phenotype were counted once and classified by the most severe phenotype observed.…”

“…Consequently, the progression of one-carbon metabolism is disrupted by the Mtrr gt mutation as evidenced by plasma hyperhomocysteinemia [10,13] and widespread changes in DNA methylation patterns [10,16,17]. Additionally, Mtrr gt/gt mice display several phenotypes similar to the clinical features of folate deficiency in humans [18] or human MTRR mutations [19,20] including macrocytic anemia [21] and neural tube closure defects (NTDs) [10,22]. Beyond this, other phenotypes have emerged in Mtrr gt/gt mice reflecting a broader influence of impaired one-carbon metabolism on development.…”

“…Beyond this, other phenotypes have emerged in Mtrr gt/gt mice reflecting a broader influence of impaired one-carbon metabolism on development. These phenotypes include fetal growth defects (such as fetal growth restriction (FGR), fetal growth enhancement (FGE), or developmental delay) [10,23], complications during implantation (e.g., twinning, skewed implantation) [10,22], haemorrhages, and/or congenital malformations (such as congenital heart defects and poor placentation) [10,22,24]. Therefore, the Mtrr gt mouse model is ideal for exploring the molecular consequences of defective one-carbon metabolism during growth and development.…”

One-carbon metabolism is required for epigenetic stability in the mouse placenta

“…Consequently, the progression of one-carbon metabolism is disrupted by the Mtrr gt mutation as evidenced by plasma hyperhomocysteinemia ( Elmore et al, 2007 ; Padmanabhan et al, 2013 ) and widespread changes in DNA methylation patterns ( Padmanabhan et al, 2013 ; Bertozzi et al, 2021 ; Blake et al, 2021 ). Additionally, Mtrr gt/gt mice display several phenotypes similar to the clinical features of folate deficiency in humans ( Krishnaswamy and Madhavan Nair, 2001 ) or human MTRR mutations ( Schuh et al, 1984 ; Wilson et al, 1999 ) including macrocytic anemia ( Padmanabhan et al, 2018 ) and neural tube closure defects (NTDs) ( Padmanabhan et al, 2013 ; Wilkinson et al, 2021 ). Beyond this, other phenotypes have emerged in Mtrr gt/gt mice reflecting a broader influence of impaired one-carbon metabolism on development.…”