Regulation Of Embryo Implantation And Its Simulation In Vitro

The content can be divided into two parts: one is about the regulation of cytokines on development and implantation of early embryos (chapter 3); the other deals with the effects of extracellular matrix on trophoblast differentiation and invasion (chapter 4&5). Accordingly, the abstract also has two parts.Abstract 1: Leukemia inhibitory factor (LIF) has been shown to play an important role in the development and implantation of blastocysts in mice. In the current study, the reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the expression patterns of LIF and its receptor (LIFR) genes in rabbit embryos during preimplantation development, and the uterine expression of LIF and LIFR was also evaluated by Western blotting. Transcripts for LIFR were detected within morula and blastocyst-stage embryos, while the LIF mRNA was only found in blastocysts (from early to fully expanded blastocoel cavities), indicating that embryo-derived LIF can act in an autocrine manner on the process of blastocyst formation. The expression levels of LIF and LIFR in uterine epithelium were gradually increased during preimplantation period and reached their highest levels on days 6.5 of pregnancy, just before the time of blastocyst implantation, suggest that paracrine LIF circuit should exist between the endometrium and the early embryos, which may be involved in the embryo-maternal dialogue and important for the blastocyst implantation. The data present here show the stage-specific and dynamic expression patterns of LIF and LIFR, both in embryos and endometrium, during early pregnancy in rabbits, which indicated that LIF might play an important role in the preimplantation development and subsequent implantation of rabbit embryos.Abstract 2: Implantation hinges on the ability of the embryo to degrade the basement membrane of uterine epithelium and to invade into the uterine stroma. This task is partly accomplished by trophoblast cells secreting proteinases capable of degrading the components of basement membrane and underlying extracellular matrix (ECM). We have utilized trophoblast stem (TS) cell line to study trophoblast differentiaition, invasion and trophoblast-ECM interactions in vitro. TS cell lines has been established from blastocysts or early postimplantation embryos. They can be maintained in a proliferative state in the presence of primary mouse embryonic fibroblast-conditioned medium (MEF-CM) and fibroblast growth factor-4 (FGF-4) , and differentiate into various trophoblast lineages when these factors are deprived. The invasive behavior of the differentiating TS cells was demonstrated using Matrigel invasion chambers, which parallels the upregulation of matrix metalloproteinase (MMP)-9 and Mmp-14. Expression of marker genes indicates that the invasive cells appeared to be mainly trophoblast giant cells, and a small portion of spongiotrophoblast cells. Striking variations in morphology and gene expression have been found when the cells were cultured on Matrigel, a rich resource of ECM proteins. The differentiated TS cells showed a highly invasive capacity on Matrigel. Under stem cell culture conditions, Matrigel promoted the differentiation of TS cells, preferably induced the formation of spongiotrophoblast, and upregulated the expression of Mmp-9 and its inhibitor Timp-3. In conclusion, TS cells exhibit invasive capacity when they differentiate into giant cells and spongiotrophoblast in vitro, which is consistent with the upregulation of Mmp-9 and Mmp-14. Differentiating TS cells invade into ECM in a three-dimensional culture, while ECM can induce the differentiation of TS cells, and upregulate the expression of Mmp-9 and Timp-3. These findings implicate that TS cell culture system can be used as a model for studying the regulation of trophoblast differentiation and invasion in vitro. In addition, we have investigated the effects of MEF-CM and FGF-4 on TS cell differentiation respectively. The results showed that, MEF-CM played a dominant role in the regulation of TS cell differentiation, and FGF-4 alone could not maintain TS cells in the stem cell state. The effects of MEF-CM on TS cells were unique, andthe unknown factors in it should be a or several protein(s).