Massive dysregulation of genes involved in cell signaling and placental development in cloned cattle conceptus and maternal endometrium

Abstract

A major unresolved issue in the cloning of mammals by somatic cell nuclear transfer (SCNT) is the mechanism by which the process fails after embryos are transferred to the uterus of recipients before or during the implantation window. We investigated this problem by using RNA sequencing (RNA-seq) to compare the transcriptomes in cattle conceptuses produced by SCNT and artificial insemination (AI) at day (d) 18 (preimplantation) and d 34 (postimplantation) of gestation. In addition, endometrium was profiled to identify the communication pathways that might be affected by the presence of a cloned conceptus, ultimately leading to mortality before or during the implantation window. At d 18, the effects on the transcriptome associated with SCNT were massive, involving more than 5,000 differentially expressed genes (DEGs). Among them are 121 genes that have embryonic lethal phenotypes in mice, cause defects in trophoblast and placental development, and/or affect conceptus survival in mice. In endometria at d 18, <0.4% of expressed genes were affected by the presence of a cloned conceptus, whereas at d 34, ∼36% and <0.7% of genes were differentially expressed in intercaruncular and caruncular tissues, respectively. Functional analysis of DEGs in placental and endometrial tissues suggests a major disruption of signaling between the cloned conceptus and the endometrium, particularly the intercaruncular tissue. Our results support a "bottleneck" model for cloned conceptus survival during the periimplantation period determined by gene expression levels in extraembryonic tissues and the endometrial response to altered signaling from clones.

Keywords: conceptus; conceptus–maternal communication; placentation; somatic cell nuclear transfer.

Fig. 1.

Representative images of the collected samples.

Fig. 2.

Genes expressed in EET during the periimplantation period: (A) overlap of genes expressed in each tissue in AI- and SCNT-derived conceptuses and (B) overlap of differentially expressed genes between SCNT and AI samples; (C) unsupervised clustering of all allantois samples collected from conceptuses. Color code for sample dendrogram: red, d-18 EET; green, d-34 chorion (CHO); and blue, d-34 allantois (ALL). Asterisks indicate SCNT samples. Expression data presented as Log2(RPKM + 1). The numbers on the nodes are the bootstrap probabilities (as percentage) of cluster certainty.

Fig. 3.

Genes expressed in endometrial tissues: (A) clustering of the endometrial samples and (B) overlap of DEGs between SCNT and AI groups for each tissue and time point. Color code for dendrogram: light blue, d-18 intercaruncular tissue (ICAR); yellow, d-18 caruncular tissue (CAR); dark blue, d-34 intercaruncular tissue; and green, d-34 caruncular tissue. Asterisks indicate SCNT samples. The numbers on the nodes are the bootstrap probabilities of cluster certainty (as percentage).

Fig. 4.

Gene network models of dysregulated ligand–receptor interactions between EETs and endometrium. (A) DEGs in d-18 SCNT EET that encode ligand proteins (above line) and putative receptors expressed in the EET and endometrial tissues (below line). (B) DEGs in d-34 endometrium from SCNT pregnancies that encode ligand proteins (above line) and putative receptors in chorion, intercaruncular, and caruncular tissues. The circle at the upper right corner of each object identifies the direction of differential expression. Blue indicates down-regulated and red up-regulated in SCNT samples compared with AI controls. All networks were created from protein interactome data. Ortholog mapping of cattle genes to human or mouse gene identifiers may have led to the appearance of unofficial gene symbols in the figure (e.g., IFNW corresponds to IFNT in cattle). Datasets S23 and S25 provide official gene symbols of the encoded proteins, National Center of Biotechnology Information gene identifiers, gene expression data, and description of potential interactions between proteins.

Fig. 5.

Hypothetical model of most representative factors for conceptus survival and pathological deficiencies in SCNT-derived pregnancies during the periimplantation period. AME, allantoic mesenchyme; AMO, allantoic mesoderm; BV, blood vessel; CAR, caruncular tissue; EMO, extraembryonic mesoderm; ICAR, intercaruncular tissue; LE, luminal epithelium; PE, parietal endoderm; TR, trophoblast.