# MTHFR as a Novel Candidate Marker for Litter Size in Rabbits

## TL;DR

This study identifies the MTHFR gene as a potential marker for predicting litter size in rabbits, based on its effects on granulosa cells and genetic variants.

## Contribution

The study discovers a novel association between the MTHFR gene and rabbit litter size, particularly the CC genotype of the g.-680C>A variant.

## Key findings

- Overexpression of MTHFR promotes apoptosis and inhibits granulosa cell proliferation.
- The CC genotype of the g.-680C>A variant is significantly associated with larger total and alive litter sizes.
- MTHFR regulates the expression of follicle-related genes like TIMP1, CITED1, and FSHR.

## Abstract

Litter size is a crucial trait in animal reproduction. This study investigated the expression of the follicle-related gene methylenetetrahydrofolate reductase (MTHFR) in high- and low-fertility groups, together with the effects of the MTHFR expression level on ovarian granulosa cells and its association with litter size. The results indicated that the overexpression of MTHFR promoted apoptosis and inhibited proliferation in granulosa cells, while regulating the expression of follicle-related genes. A significant association was also found between both the total and alive litter sizes and the CC genotype with the variant g.-680C>A in the core promoter region of MTHFR. These findings suggest that the CC genotype could serve as a useful breeding marker in rabbits.

Litter size is a significant economic trait during animal reproduction. This current study attempted to decipher whether MTHFR promotes the apoptosis of granulosa cells (GCs) and inhibits their proliferation by investigating the effects of the MTHFR gene using flow cytometry and a Cell Counting Kit-8 (CCK-8) assay. MTHFR is linked with ovarian follicle development in the reproductive performance of 104 female New Zealand rabbits. We observed that MTHFR could regulate the mRNA of follicular development-related genes (TIMP1, CITED1, FSHR, GHR, HSD17B1, and STAR) with a qRT-PCR, and we observed the protein expression of CITED1 and GHR using a western blot (WB) analysis. The dual luciferase activity assays helped identify the core promoter region of the MTHFR gene, and the polymorphism of the MTHFR promoter region was studied using Sanger sequencing. The results indicated four single nucleotide polymorphisms (SNPs) within the core promoter region, among which the g.-680C>A locus was significantly associated with both the total and alive litter sizes. Additionally, the CC genotype was associated with the largest total and alive litter sizes, compared to the CA and AA genotypes (p < 0.05). In conclusion, this study investigated the effects of MTHFR on ovarian granulosa cells and its association with selected reproductive parameters in rabbits. The results provide a theoretical foundation for the use of MTHFR as a molecular marker in rabbits.