Evaluation of Vascular Endothelial Growth Factor Gene Expression in Recellularized Liver Tissue by Mouse Embryo Fibroblast.

Background: The aim of the present study was to evaluate alterations in the vegf gene expression as an angiogenic factor in mouse embryo fibroblasts seeded on the decellularized liver fragments. Methods: Liver tissue samples (n = 10) collected from adult male mice were randomly divided into decellularized and native control groups. Tissues were decellularized by treating with 1% Triton X-100 and 0.1% SDS for 24 hours and assessed by H&E staining and SEM. Then DNA content analysis and toxicity tests were performed. By centrifugation, DiI-labeled mouse embryo fibroblasts were seeded on each scaffold and cultured for one week. The recellularized scaffolds were studied by H&E staining, SEM, and LSCM. After RNA extraction and cDNA synthesis, the expression of the vegf gene in these samples was investigated using real-time RT-PCR. Results: Our observations showed that the decellularized tissues had morphology and porous structure similar to the control group, and their DNA content significantly reduced (p < 0.05) and reached to 4.12% of the control group. The MTT test indicated no significant cellular toxicity for the decellularized scaffolds. Light microscopy, SEM, and LSCM observations confirmed the attachment and penetration of embryonic fibroblast cells on the surface and into different depths of the scaffolds. There was no statistically significant difference in terms of vegf gene expression in the cultured cells in the presence and absence of a scaffold. Conclusion: The reconstructed scaffold had no effect on vegf gene expression. Decellularized mouse liver tissue recellularized by embryonic fibroblasts could have an application in regenerative medicine.

The Effect of Sodium Selenite on Expression of Mitochondrial Transcription Factor A during In Vitro Maturation of Mouse Oocyte.

BACKGROUND: The aim of the present study was to investigate the effect of Sodium Selenite (SS) supplemented media on oocyte maturation, expression of mitochondrial transcription factor A (TFAM) and embryo quality. METHODS: Mouse Germinal Vesicle (GV) oocytes were collected after administration of Pregnant Mare Serum Gonadotropin (PMSG); in experimental group 1, oocytes were cultured and then subjected for in vitro maturation in the absence of SS, and in experimental group 2, they were matured in vitro in the presence of 10 ng/ml of SS up to 16 hr. The control group included MII oocytes obtained from the fallopian tubes after ovarian stimulation with PMSG, followed by human chorionic gonadotropin. Then, the expression of TFAM in MII oocytes in all three groups was investigated using real-time RT-PCR. The fertilization and embryo developmental rates were assessed, and finally the quality of the blastocysts was evaluated using propidium iodide staining. RESULTS: The oocyte maturation rate to MII stage in SS treated group was significantly higher than non-treated oocytes (75.65 vs. 68.17%, p<0.05). Also, the rates of fertilization, embryo development to blastocyst stage as well as the cell number of blastocyst in SS supplemented group were higher than other experimental group (p<0.05). There was a significant decrease in TFAM gene expression in both in vitro groups compared to the group with in vivo obtained oocytes (p<0.05). Moreover, there was a significant increase in TFAM gene expression in oocytes that matured in the presence of SS compared to that of the group without SS (p<0.05). CONCLUSION: Supplementation of oocyte maturation culture media with SS improved the development rate of oocytes and embryo and also enhanced TFAM expression in MII oocytes which can affect the mitochondrial biogenesis of oocytes.

Apoptosis of human ovarian tissue is not increased by either vitrification or rapid cooling.

This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in vitrified and rapidly cooled human ovarian tissue. Apoptosis was assessed 30 min and 24h after warming using transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation, as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryoprotectant solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis and either protocol could be an alternative to slow cooling of ovarian tissue. This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in human ovarian tissue cryopreserved using two different methods, i.e. vitrification and rapid cooling. Apoptosis was assessed in tissue 30 min and 24h after warming using transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryopreservation solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis in human ovarian tissue and either protocol could be an alternative to slow cooling for the preservation of ovarian tissue.

Effect of ovarian stimulation on the endometrial apoptosis at implantation period.

BACKGROUND: Apoptosis is a process that plays an important role during early stage of implantation. The aim of this study was to investigate the incidence of apoptosis in mice endometrium after ovarian stimulation at implantation period. METHODS: NMRI female mice were divided into two groups: 1) control group, which were rendered pseudopregnant by vaginal stimulation and 2) experimental group, which were stimulated using an intrapritoneal injection of 10 IU hMG followed by another injection of 10 IU hCG after 48 h. In the evening of the second injection, the mice were rendered pseudopregnant the same as control group. Samples were obtained from 1/3 middle part of uterine horns during implantation period. Apoptosis was assessed in two groups at implantation period using light and electron microscopic studies, TUNEL staining and semiquantitative RT-PCR. RESULTS: Our morphological and ultrastructural results showed apoptosis in both groups, while TUNEL analysis showed that the percentage of TUNEL-positive cells was higher in stimulated group than in the control group (P≤0.05). The expression of P53, Fas and FasL mRNA was similar in two groups but Bax and Bcl2 were much higher in control group than in the stimulated group (P≤0.05). The ratio of Bax/Bcl2 expression was much higher in stimulated group than in the control group (P≤0.05). CONCLUSION: The ovarian stimulation could change the expression of some apoptosis-related genes and enhance the incidence of endometrial apoptosis at implantation period; thus, it could affect on the implantation rate and endometrial receptivity.

Analysis of apoptosis and expression of genes related to apoptosis in cultures of follicles derived from vitrified and non-vitrified ovaries.

The aim of this study was to evaluate the incidence of apoptosis after in vitro culture of isolated follicles derived from vitrified and non-vitrified ovaries. Mouse ovaries were vitrified and their pre-antral follicles were mechanically isolated and cultured for 10 days. Growth and survival rates of the follicles were assessed during the culture period and the ultrastructure of the follicles was studied. The expression of p53, Bcl-2, Bax, Fas, FasL and survivin were analyzed by real-time RT-PCR in different follicular developmental stages. The percentages of apoptotic and necrotic cells were determined using a fluorescein-activated cell sorting (FACS) technique. There were no differences between the growth and survival rates of follicles in the vitrified and non-vitrified groups. All of the evaluated genes were expressed in the pre-antral, large pre-antral and antral follicles in both groups, except Fas mRNA, which was not expressed in the pre-antral follicles. The expression of p53, Bcl2, Bax and FasL mRNA was similar in vitrified and non-vitrified groups; however, Fas mRNAs were more strongly expressed in the antral follicles of the vitrified group than of the control group (P < 0.05). The expression of survivin 140 was lower in the antral follicles of the vitrified group than of the control group (P < 0.05). FACS analysis showed that the percentage of intact cells was lower in the vitrified group than in the non-vitrified group (P < 0.05). This study demonstrated no signs of apoptosis ultrastructurally in cultured follicles; however, vitrification was shown to affect the expression of some genes related to apoptosis.