Aberrant gene expression patterns in placentomes are associated with phenotypically normal and abnormal cattle cloned by somatic cell nuclear transfer

Abstract

Transcription profiling of placentomes derived from somatic cell nuclear transfer (SCNT, n = 20), in vitro fertilization (IVF, n = 9), and artificial insemination (AI, n = 9) at or near term development was performed to better understand why SCNT and IVF often result in placental defects, hydrops, and large offspring syndrome (LOS). Multivariate analysis of variance was used to distinguish the effects of SCNT, IVF, and AI on gene expression, taking into account the effects of parturition (term or preterm), sex of fetus, breed of dam, breed of fetus, and pathological finding in the offspring (hydrops, normal, or other abnormalities). Differential expression of 20 physiologically important genes was confirmed with quantitative PCR. The largest effect on placentome gene expression was attributable to whether placentas were collected at term or preterm (i.e., whether the collection was because of disease or to obtain stage-matched controls) followed by placentome source (AI, IVF, or SCNT). Gene expression in SCNT placentomes was dramatically different from AI (n = 336 genes; 276 >2-fold) and from IVF (n = 733 genes; 162 >2-fold) placentomes. Functional analysis of differentially expressed genes (DEG) showed that IVF has significant effects on genes associated with cellular metabolism. In contrast, DEG associated with SCNT are involved in multiple pathways, including cell cycle, cell death, and gene expression. Many DEG were shared between the gene lists for IVF and SCNT comparisons, suggesting that common pathways are affected by the embryo culture methods used for IVF and SCNT. However, the many unique gene functions and pathways affected by SCNT suggest that cloned fetuses may be starved and accumulating toxic wastes due to placental insufficiency caused by reprogramming errors. Many of these genes are candidates for hydrops and LOS.

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

“…Our group has investigated the effects of SCNT cloning on gene expression and on developmental anomalies at different phases of cattle development and in different tissues, ranging from day (d) 7 blastocysts to placentomes that were collected at the term of pregnancy (14,(17)(18)(19). The resemblance of gene expression profiles between SCNT-derived blastocysts and those derived by artificial insemination (AI) (18) was the first evidence that the effects of cloning by SCNT could be more severe after blastocyst hatching.…”

“…At d 18, the beginning of the implantation window, developmental asynchrony between the embryonic disk and the extraembryonic tissue (EET) was shown to be one reason for arrest at implantation (19). In addition, relatively few differentially expressed genes are detected in cloned fetuses (20,21), and comparison of gene expression in term placentomes collected from pregnancies produced by SNCT, in vitro fertilization, and AI (17) showed that, at later stages of development and at term, placentomes of SCNT-derived pregnancies had an altered transcriptome, regardless of whether pathological morphology was observed (15,17). These studies demonstrated that the abnormal development of the placenta is the major cause of failure of clones to complete pregnancy after implantation.…”

“…Thus, multiple lines of experimental evidence have led to the conclusion that EET is the origin of the developmental pathologies in placentation of SCNTderived clones (17,(22)(23)(24)(25)(26). Therefore, study of the communication between cloned conceptuses and the endometrium should yield important insights into the mechanisms underlying average losses of ∼60% of cloned embryos before implantation and 80% of fetuses within the first trimester of gestation (27,28).…”

“…These bottlenecks would eliminate most abnormal clones, but some placentas may compensate enough for few clone pregnancies to progress to term despite morphological and physiological abnormalities (17). Thus, even though there are a relatively small number of dysregulated genes at d 34 in EETs and endometrium in SCNT-derived gestations, the numbers of DEGs may again increase as the placenta develops further and additional molecular and cellular functions are required (17).…”

“…Embryonic and fetal losses substantially reduce the efficiency of SCNT and thus present a significant limitation to the widespread application of the technology for animal improvement. However, the disrupted embryonic and fetal development of cattle clones produced by SCNT has been used as a model to elucidate the mechanisms of embryo loss, the maternal recognition of pregnancy (13,14), and placental development (15)(16)(17). Understanding the mechanism of embryonic and fetal losses in SCNT clones is thus not only practically important but also serves as a window to understanding infertility in cattle and other mammals.…”