The Reconstruction Of The 3D Rabbit Uterine Tissue Patch And Its Effect On The Development Of Mouse Embryos In Vitro

Uterus is the important reproductive organ for female animals. The studies about the pathology and physiology of uterus can promote the development of the development biology and reproductive medicine. Uterus changes dynamically in morphology and physiology with the estrous cycle of female animals, this made it difficult to study the uterus in vivo. This study want to isolate and culture the uterine cells of the pseudpregnant rabbit as seeding cells and use the typeⅠcollagen of mouse tail as scaffolding to regenerate the three-dimensional (3D) uterine tissue patch containing the epithelial layer, stromal layer and smooth muscle layer according to the native structure. The regenerated 3D uterine patch may be used as an in vitro model for the study of pathology and physiology of uterus. The 2-cell mouse embryos or mouse blastula were co-cultured with this 3D uterine patch to study its effect on the development and the implantation of the embryos, so we can present an in vitro model for the study of development biology. The main results showed as follows:1.The rabbit endometrial epithelial cells and stromal cells of the pseudpregnant rabbit can be cultured and proliferated in vitro by tissue digested using 0.1% collagenase. The high purity epithelial cells and stromal cells were isolated using centrifugation method. 100 nmol/Lβ-estradiol (E2) and 10 nmol/L progesterone (P4) in media can stimulate the proliferation of epithelial cells, and 100 nmol/L P4 in media can stimulate the proliferation of stromal cells (P<0.05). Immunocytochemistry staining demonstrated that cytokeratin was positive in epithelial cells and vimentin was detectable in stromal cells. The stromal cells can be subcultured for 6-7 passages and the epithelial cells can be subcultured for 4-5 passages when cocultured with 15% stromal cells in media containing 100 nmol/L E2 and 10 nmol/L P4. This study demonstrated that sex steroids can promote proliferation of endometrial cells in vitro and maintain its specificity and homogeneity. The culture and proliferation of endometrial cells can provide enough seeding cells for the regeneration of 3D uterine tissue in vitro.2.The smooth muscle cells of uterus can be prepared by tissue cutting and 0.25% trypsin solution digested. 100 nmol/L E2 in media can stimulate the proliferation of smooth muscle cells, but 100 nmol/L P4 in media can inhibit the proliferation of smooth muscle cells(P< 0.05). Immunocytochemistry staining demonstrated thatα-smooth muscle actin was positive in smooth muscle cells. The smooth muscle cells can be subcultured for 9-10 passages in vitro. The culture of uterine smooth muscle cells can facilitate the elucidation of physiology of uterine smooth muscle and provide the enough seeding cells for the regeneration of 3D uterine tissue in vitro.3.The stromal cells and smooth muscle cells prepared in vitro were inoculated respectively into liquid typeⅠcollagen derived from mouse tails and Matrigel, then the epithelial cells were cultured on its surface to reconstruct uterine 3D tissue in vitro. Using this method we successfully reconstruct the 3D uterine tissue in vitro. H.E. staining indicate 3 layers structure of 3D uterine tissue. Immunohistochemistry staining of anti-cytokeratin, anti- vimentin and anti-α-smooth muscle actin showed that the 3 layers of 3D uterine tissue are formed by the epithelial cells, stromal cells and smooth muscle cells respectively. Pinopodes, microvilli and cilia on the surface of epithelium are demonstrated by scanning electron microscope.Tight junction and desmosomes between luminal epitheliums are observed by transmission electron microscope(TEM). The abundant mitochondrion were also observed in the cytoplasm by TEM. Expression of Hoxa-10, integrinβ3 and HB-EGF genes are detected in the reconstructed uterus respectively after stimulated by sex steroids, but not expressed or expressed weakly in the 3D uterine tissue not stimulated by sex. These results showed that the regenerated rabbit 3D uterine tissue have some common features in the structure and physiology compared with the native uterus. The 3D uterine tissue patch can be used as a ideal model for the uterine physiology and pathology in vitro.4.The 2-cell embryos of mouse were co-cultured with the 3D uterine tissue in the 12 well plate to establishing an effective co-culture system to improve the development rate and quality of early embryos in vitro. The results indicate that more embryos in the 3D co-culture system reached the blastocyst stage than that in simple CZB media cultere system and in one layer reproductive tract cell co-culture system; 3D co-culture system can increase the hatching rate of the blastocysts and the attachment rate of hatched blastocysts compared to 3D co-culture system. 3D co-culture system can significantly increase the cell numbers of the blastocyst compared to the other culture system。The conclusions is that the 3D co-culture system can better simulate the natural developing environment of embryos in vitro and improve the development rate and quality of early embryos.5.The blastula of mouse were co-cultured with the 3D uterine tissue to establish a new model for the study of the embryos implantation. The reconstructed 3D uterine tissue was cultured in CZB media containing 100 nmol/L progesterone, 10 nmol/Lβ-estradiol for 3 days. Then the media were replaced with CZB containing 100 nmol/L progesterone, 10 nmol/L β-estradiol, and the mouse blastula were co-cultured with it. The results showed that the blastula shedding from zona pellucida and attach to the surface of 3D uterine tissue patch, then penetrate the epithelial layer and implanted into the stromal layer. The microtube-like structure was observed in the developing embryos implanted into the 3D uterine tissue. The reconstructed 3D uterine tissue can serve as a robust model for the embryo implantation and the differentiation of the inner cell mass of blastula in vitro.