Materials and Methods: Limbal biopsies were taken from normal human limbus (Central Eye Bank of Iran), and were cultured as an explant on amniotic membrane and plastic dish. Cells were exposed to the air after 14 days of culture. The expression of proposed limbal stem cells markers (p63, ABCG2), corneal markers (K3/12, Connexin43) and also HSPs (90, hsc70) were analyzed by RT-PCR, immunocytochemistry and flowcytometry pre and post air exposure at day 14 (before air exposure) and 30 (after air exposure) respectively.Results: ABCG2 positive cells with high expression of p63 and low expression of K3/K12 and Connexin43 were considered as limbal stem cells while ABCG2 negative cells with low expression of p63 and high expression of K3/K12, Connexin43 were assumed as corneal cells. Our data showed that HSPs expressed strongly at mRNA level before and after air exposure while they expressed in different pattern at protein level. It was interested that HSP90 proteins were expressed weakly in limbal stem cells at day 14 on both groups, but its expression increased after air exposure at day 30 (p<0.05) only in AM group on the other hand HSC70 were expressed strongly on plastic dish in compared with amniotic membrane (p<0.05) before air exposure but after air exposure its expression has significant increasing in AM group.Conclusion: We assumed hsp90 has important role during differentiation process but only in early stage of differentiation because its increasing was in AM group,and in this group we had TAC cells(early differentiated cells) after air exposure but Hsc70 expression may related with differentiation process in both early and terminally stage of differentiation in cultured limbal stem cell.

Objective: The aim of this study was to investigate the effects of two vitrification protocols on mouse metaphase stage II (MII) oocytes and evaluate their effects on the expression of heat shock protein A1A (HSP A1A) and MnSOD genes.Materials and Methods: Groups of approximately 15 oocytes without cumulus complexes were collected and vitrified with 10% (v/v) ethylene glycol (EG) + 10% (v/v) dimethylsulphoxide (DMSO) + 0.5M sucrose in group A (VSI) and 14.5% (v/v) EG + 14.5% PROH + 0.5M sucrose in group B (VSII), respectively. Thawing after vitrification was performd by placing the oocytes into 1M sucrose for 1 minute and two diluted solutions, each for 3 minutes. After thawing, the oocytes were fertilized and cultured in vitro to develop into the pronuclear stage. The survival rate of vitrified-warmed oocytes and rate of fertilization were evaluated. In addition, gene expressions (HSP A1A, MnSOD and b actin) of vitrifiedwarmed oocytes were also examined by reverse transcription polymerase chain reaction (RT-PCR).Results: Survival rates of each group were separately compared to the control. The result showed significant differences between each experimental group compared to the control (p£0.001). The survival rate of oocytes after warming was higher in VSI (91.2% ± 1.7) compared to VSII (89.2% ±1.5) but there were no significant differences between the two groups. The rate of fertilization was significantly (p£0.05) reduced in vitrified-warmed (VSI: 39% ± 5.8; VSII: 34% ± 5.7) oocytes compared to the control (88.36% ± 2.3). The expression of MnSOD increased in the vitrified-warmed oocytes when compared to control oocytes. We also detected HSP A1A only in the control and VSI group.Discussion: Vitrification of oocytes by cryotop resulted in high survival rate; low developmental competence and fertilization rate of vitrified-warmed oocytes which may be a result of changingexpression of important genes after thawing.