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

Abstract

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.

In vitro maturation, apoptotic gene expression and incidence of numerical chromosomal abnormalities following cryotop vitrification of sheep cumulus-oocyte complexes

“…It seems likely that cell death in the cryotop vitrification procedure has occurred through another pathway. In agreement with mentioned results, some researchers showed that the expression of apoptotic genes in the follicles derived from vitrified-warmed ovarian tissue strips were similar to the control group and no sign of ultrastructurally apoptosis were seen in the vitrified follicles [45]. Additionally, Anchamparuthy et al [46] demonstrated that transcript levels of Fas, FasL, Bax and Bcl-2 increased post vitrification-warming and maturation of germinal vesicle oocytes but despite of their prospect any evidence of related apoptosis were not observed in embryos derived from large numbers of vitrified oocytes.…”

New pyrazolo‐[3,4‐ d ]‐pyrimidine derivative Src kinase inhibitors lead to cell cycle arrest and tumor growth reduction of human medulloblastoma cells

“…Bax also forms heterodimers with Bcl2 and counteracts its death‐repressor activity. Given that Bax and Bcl2 play an important role in apoptosis (39), we verified the time course effects of the compounds on their expression in Daoy cells. We observed a time‐dependent increase in Bax protein levels starting from 48 h of treatment and, at the same time, a decrease of Bcl2 levels in S7‐ and SI163‐treated cells.…”

In vitro development of human primordial follicles to preantral stage after vitrification

“…They concluded that cryopreservation caused the death of ovarian cells through apoptosis and thus inhibiting further development of primordial follicles [61]. However a study by Mazoochi et al(2008) [62] carried out in the mouse showed that there was no sign of apoptosis in vitrified and cultured follicles [62]. A study carried out with human ovarian tissue also reported that vitrification has no effect on apoptosis of follicles [63,64].…”