Maternal Undernutrition Affects Fetal Thymus DNA Methylation, Gene Expression, and, Thereby, Metabolism and Immunopoiesis in Wagyu (Japanese Black) Cattle.

We aimed to determine the effects of maternal nutrient restriction (MNR) on the DNA methylation and gene expression patterns associated with metabolism and immunopoiesis in the thymuses of fetal Wagyu cattle. Pregnant cows were allocated to two groups: a low-nutrition (LN; 60% nutritional requirement; n = 5) and a high-nutrition (HN; 120% nutritional requirement, n = 6) group, until 8.5 months of gestation. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing were used to analyze DNA methylation and gene expression, while capillary electrophoresis-Fourier transform mass spectrometry assessed the metabolome. WGBS identified 4566 hypomethylated and 4303 hypermethylated genes in the LN group, with the intergenic regions most frequently being methylated. Pathway analysis linked hypoDMGs to Ras signaling, while hyperDMGs were associated with Hippo signaling. RNA sequencing found 94 differentially expressed genes (66 upregulated, 28 downregulated) in the LN group. The upregulated genes were tied to metabolic pathways and oxidative phosphorylation; the downregulated genes were linked to natural killer cell cytotoxicity. Key overlapping genes (GRIA1, CACNA1D, SCL25A4) were involved in cAMP signaling. The metabolomic analysis indicated an altered amino acid metabolism in the MNR fetuses. These findings suggest that MNR affects DNA methylation, gene expression, and the amino acid metabolism, impacting immune system regulation during fetal thymus development in Wagyu cattle.

Gene expression of bovine endometrial epithelial cells cultured in matrigel.

Glandular epithelial cells (GE) in the endometrium are thought to support the elongation and survival of ruminant embryos by secreting histotrophs. In the present study, the gene expression of bovine endometrial epithelial cells cultured in matrigel was analyzed and examined whether it could be an in vitro model of GE. Bovine endometrial epithelial cells (BEE) and stromal cells (BES) were isolated from the slaughterhouse uteri and cultured in DMEM/F12 + 10% FBS. BEE showed the gland-like structure morphological changes when cultured in 15% matrigel but could not be identified in higher concentrations of the matrigel (30% or 60%). The expression of typical genes expressed in GE, SERPINA14 and GRP, was substantially high in matrigel-cultured BEE than in monolayer (P  <  0.05). P4 and INFα have no significant effect on the SERPINA14 expression of BEE cultured in matrigel without co-culture with BES. On the other hand, when BEE were co-cultured with BES in matrigel culture, the expression of FGF13 was increased by the P4 treatment (P  <  0.05). Furthermore, SERPINA14 and TXN expressions were increased by P4 + IFNα treatment (P  <  0.05). These results demonstrate the appropriate conditions for BEE to form glandular structures in matrigel and the effect of co-culture with BES. The present study highlighted the possible use of matrigel for the culture of BEE to investigate the expression of cell-specific glandular epithelial genes as well as P4 and type-I IFN as factors controlling endometrial function during the implantation period.

Analysis of novel embryonic factors of cattle and effects on endometrial cells in vitro.

Interferon tau (IFNT) is thought to have essential functions in maternal recognition and establishment of pregnancy in ruminants. There, however, is a lack of research on embryonic factors that affect pregnancy other than IFNT. The present study was conducted to determine what are other embryo-derived factors involved in pregnancy recognition and to identify effects on endometrial cells using an in vitro culture system. With use of LC-MS/MS procedures to evaluate the supernatant of elongating embryos of cattle in culture, there was detection of 78 secretary proteins including five cytokines and two growth factors. Then there was analysis for up-regulated genes using ingenuity pathway procedure, IFNT and MIF were identified as upstream regulators of 37 and five genes, respectively. The mRNA transcript of MIF receptors was identified in endometrial cells, however, not in embryos. Among genes induced by MIF, CCL2, IL7 and IL23A transcripts were identified in endometrial cells. When endometrial cells were treated with interferon alpha (IFNA) and MIF, the CCL2 transcript was in a larger abundance of endometrial epithelial and polymorphonuclear neutrophil cells, and there was a larger abundance of there mRNA transcripts as a result of MIF treatment of peripheral blood mononuclear cells. In conclusion, MIF secreted by elongating embryos of cattle synergistically regulates relative abundances of specific mRNA transcripts of endometrial cells when there is treatment with IFNA, indicating further there are several factors other than IFNT that have effects on gene expression in the endometrium during early stages of gestation in cattle.