| Thymosin-β4 (thymosin-β4, Tb4) is a small hydrophilic peptide, initially separated and purified from bovine thymus componentⅤ, which molecular weight is about 5 k Da, and composed of 43 amino acid residues. Current researches had showed that Tb4 is an important G-actin sequestering factor in cells, which could regular the activity of G-actin in various microfilaments events. Tb4 molecule is highly conservative, which has been founded in a variety of tissues and organs in vertebrates, and also exists in part invertebrates. This peptide has extensive biological functions, recent studies have shown that it closely relates with many physiological and pathological activities, such as immune regulation, neurological development, wound healing, inflammatory response, angiogenesis, apoptosis, tumor occurrence and migration, etc. Previously, many researches have reported that Tb4 has broad cellular and physiological functions, but until now, there is lack of systematic research on mammal oocytes and early embryonic development.In the research, we studied with the ovary, oocytes and early embryos of Kunming mouse. During the experiment, the immuno-histochemistry, immuno-fluorescence histochemistry and RT-PCR technique were utilized as chief study methods. The discipline of Tb4 expression and distribution in oocytes maturation and early embryonic development were studied both in vivo and vitro. In this experiment we found that, there exist different locative expressions between early embryonic cells, which of different developmental phases. Among those stages, Tb4 have a nucleus and cytoplasm type of distribution. So, at the same time, we used of mouse embryonic fibroblasts (MEFs) to test the phenomenon from MEFs proliferation and inhibited in specific cell cycle experiment.The results showed as following:1. Mouse ovarian immuno-histochemistry results showed that, during follicle development, Tb4 protein expression gradually increased in the oocytes. This molecule expressed weekly in nucleus and cytoplasm of primary follicle oocytes. But during later stages of follicle maturation, Tb4 was increasing in cytoplasm, particularly in the nuclear membrane, while the expression was weak in nuclear. At the same time, Tb4 also expressed weekly in granulose cells of antral follicles, and there was no distribution in the nucleus, either.2. Confocal laser scanning results showed that Tb4 protein expressed with a different location in GV stage oocytes, which divided in different groups by chromatin configuration.3. Confocal laser scanning results also showed that, Tb4 in mouse oocytes maturation and early embryonic development process, there are differences of expression and localization. Of which changes, it had relationship with oocytes maturation and early embryonic development and the specific stages of cell cycle.4. Confocal laser scanning results showed that in the early blastula implantation, Tb4 protein expressed higher in the side of implantation, revealed Tb4 could regulate the embryo implantation by actived the actin depolymerization activity processes.5. From the beginning of oocytes maturation until the formation of the early blastula implantation, Tb4 protein expression showed an increasing tendency. Particularly in the morula, blastula and early implantated blastula, Tb4 had a higher expression induced the early development of embryos to adapt the characteristics of physical activity.6. In vitro cultured early embryos, Tb4 mRNA began to increase from the zygotes stage, until the morula and blastula stages, the expression reached a peak. In this process, specially, in the 2-cells stage, this molecular expression of mRNA had take a sub-peak amount, it was significantly higher than near developmental stages.7. This experiment confirmed that a high level with microfilament polymerization or deploy- merization activity changes in cell morphology of MEFs, those can determine the distributive type of Tb4 location. In the course of MEFs proliferation, cell cycle factors also induced Tb4 positioning. The detail of it was G1-phase cells showed a high percentage of NH distribution type, but in S cell cycle it for a high percentage of type NL positioning, and after S period, NH expression type ratio gradually return to a higher level.8. The study of Tb4 found that the location type of MEFs also had a relationship to the cell proliferation density. In the same cultured system, MEFs of different colony density, it performed main different distribution. The details follow as high-density cells showed NH expression type, and the low-density region showed NL type positioning.The research concluded that Tb4 mainly through combines of G-actin monomer to sequence the activity of it, and regulate the activities of G-actin. As the physiological adjustments function to ensure that some changes in cell morphology and growth or proliferation related with microfilaments. Our research showed that, Tb4 could regulate microfilaments events (such as polymerization and depolymerization) and proliferation of cells in the developmental processes through the changes of its expression and localization. Overall, Tb4 plays an important role for mouse oocytes maturation, early embryonic development and embryos implantation.
|
PDF Full Text Request