Expression of triml2 in early conveptus development and placentation during pregnancy in the pig
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The eutherian amniote evolved to utilize the oviparous egg membranes to form a placenta which allowed the fetus to grow in utero for an extend gestational period. For this to occur, genes specific to placentation either had to be gained or lost. TRIML1 and TRIML2 are genes thought to have developed to play a role in viviparity. Tripartite Motif Family-Like Protein 1 and 2 are associated with the downregulation of inflammation. TRIML1 is preferentially expressed in the testes, while TRIML2 is selectively expressed in preimplantation embryos and trophoblast derived structures like the chorion. TRIML1 has a modified protein structure compared to the marsupial. Eutherian amniotes and marsupials are the only animals that express this protein but the functions differently in each species. TRIML2 expression is unique to eutherian species (not expressed in marsupials). TRIML1 and TRIML2 may be responsible for regulating inflammatory phase during attachment or invasion of the conceptus to establish and maintain the pregnancy to term, more specifically TRIML2. The placenta is very complex and has evolved by regulating the inflammatory phase of pregnancy during conceptus attachment and development of the placental tissues. TRIML2 is unique compared to the other 80 TRIM proteins in that it lacks both the RING and B-box domains that may adopt RING-like folds. In general TRIM proteins consist of an N-terminal RING domain followed by one or two B-box domains. The specific expression of TRIML1 and TRIML2 in the placenta and gonads suggest a possible role in the transition to viviparity in mammals. The objective of the present study was to determine the mRNA expression patterns of TRIML1 and TRIML2 during early embryo development, conceptus attachment and placental formation during pregnancy in the pig. To evaluate the tissue specificity of TRIML1 and TRIML2, mRNA was extracted from a panel of tissues which included oocytes, early embryos, conceptuses and placenta. Expression of TRIML1 and TRIML2 was detected in the blastocyst, spherical stage conceptuses, and conceptuses tissues collected on day 12, 15, 17, 21 as well as in the chorioallantois regions on day 28, 45 and 60 of gestation. TRIML1 was expressed only in the testes, while TRIML2 is expressed in the ovary and testis but absent from all the other tissues in the tissue panel. The specific and unique expression of TRIML2 in the placenta and testis is consistent with it serving as an anti-inflammatory factor to provide immunological protection of the eutherian placenta. RNAscope in-situ hybridization of indicated that TRIML1 was present in round spermatocytes within the seminiferous tubes of testis but was absent in the placenta. TRIML2 expression was localized in the trophectoderm and chorion of the conceptus and placenta respectively. CRISPR/Cas 9 gene editing with gRNA was utilized to target TRIML1 and TRIML2 expression in fetal fibroblast cells. TRIML1-/- and TRIML2-/- fibroblast cells with biallelic edits were developed for somatic cell nuclear transfer and embryos generated were transferred into surrogate gilts. Early conceptus and placental development (Day 21 and 35) were not affected by the loss of TRIML1-/- or TRIML2-/- expression.
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M.S.