Increased expression of pro-inflammatory cytokines at the fetal-maternal interface in bovine pregnancies produced by cloning

Abstract

Problem: A significant rate of spontaneous abortion is observed in cattle pregnancies produced by somatic cell nuclear transfer (SCNT). Major histocompatibility complex class I (MHC-I) proteins are abnormally expressed on the surface of trophoblast cells from SCNT conceptuses.

Method of study: MHC-I homozygous compatible (n = 9), homozygous incompatible (n = 8), and heterozygous incompatible (n = 5) pregnancies were established by SCNT. Eight control pregnancies were established by artificial insemination. Uterine and trophoblast samples were collected on day 35 ±1 of pregnancy, the expression of immune-related genes was examined by qPCR, and the expression of trophoblast microRNAs was assessed by sequencing.

Results: Compared to the control group, trophoblast from MHC-I heterozygous incompatible pregnancies expressed increased levels of CD28, CTLA4, CXCL8, IFNG, IL1A, IL2, IL10, IL12B, TBX21, and TNF, while GNLY expression was downregulated. The MHC-I homozygous incompatible treatment group expressed increased levels of IFNG, IL1A, and IL2 while the MHC-I homozygous compatible group did not differentially express any genes compared to the control group. In the endometrium, relative to the control group, MHC-I heterozygous incompatible pregnancies expressed increased levels of CD28, CTLA4, CXCL8, IFNG, IL10, IL12B, and TNF, while GATA3 expression was downregulated. The MHC-I homozygous incompatible group expressed decreased amounts of CSF2 transcripts compared with the control group but did not have abnormal expression of any other immune-related genes. MHC-I incompatible pregnancies had 40 deregulated miRNAs compared to control pregnancies and 62 deregulated microRNAs compared to MHC-I compatible pregnancies.

Conclusions: MHC-I compatibility between the dam and fetus prevented an exacerbated maternal immune response from being mounted against fetal antigens.

Keywords: cattle; cytokines; gene expression; microRNA; miscarriage; pregnancy; somatic cell nuclear transfer.

FIGURE 1

Proportional Venn diagram depicting the number of up‐ (top) and down‐ (bottom) regulated trophoblast miRNAs in day 35 ± 1 pregnancies where fetal and maternal major histocompatibility complex class I (MHC‐I) haplotypes were matched (homozygous compatible, n = 9) and unmatched (heterozygous incompatible; n = 5). Control pregnancies were established by artificial insemination (n = 8)

FIGURE 2

Log2 fold change of differentially expressed miRNAs in trophoblasts of MHC‐I heterozygous incompatible (n = 5) SCNT pregnancies compared to control pregnancies established by artificial insemination (n = 8) (A); and between MHC‐I heterozygous incompatible (n = 5) and MHC‐I homozygous compatible (n = 9) SCNT pregnancies (B) at gestation day 35 ± 1. MHC‐I, major histocompatibility complex class I; SCNT, somatic cell nuclear transfer

FIGURE 3

Results of GO analysis of predicted target genes for downregulated miRNAs (A) and upregulated miRNAs (B) between MHC‐I heterozygous incompatible (n = 5) and control trophoblast (n = 8), and predicted target genes for downregulated miRNAs (C) and upregulated miRNAs (D) between MHC‐I heterozygous incompatible (n = 5) and MHC‐I homozygous compatible (n = 9) trophoblast. Blue bars indicate molecular function, green bars indicate cellular components, and red bars indicate biological process. Day 35 ± 1 control pregnancies were established by artificial insemination and MHC‐I heterozygous incompatible and homozygous compatible pregnancies were established by somatic cell nuclear transfer. Enrichment score [−log10(p‐value)] is a measure of how strongly the gene is associated with a specific biological process. GO, Gene Ontology; MHC‐I, major histocompatibility complex class I

FIGURE 4

Pathway analysis by KEGG of predicted target genes of downregulated (A) and upregulated (B) miRNAs in bovine trophoblast of MHC‐I heterozygous incompatible (n = 5) compared with control (n = 8) pregnancies, and predicted target genes of downregulated (C) and upregulated (D) miRNAs in bovine trophoblast of MHC‐I heterozygous incompatible (n = 5) compared with MHC‐I homozygous compatible (n = 9) pregnancies at day 35 of gestation. Control pregnancies were established by artificial insemination and MHC‐I heterozygous incompatible and homozygous compatible pregnancies were established by somatic cell nuclear transfer. Enrichment score [−log10(p‐value)] is a measure of how strongly the gene is associated with a specific biological process. KEGG, Kyoto Encyclopedia of Genes and Genomes; MHC‐I, major histocompatibility complex class I