Development of mouse preantral follicle after in vitro culture in a medium containing melatonin

Improvements in cancer treatment have allowed more young women to survive. However, many cancer patients suffer from ovarian failure. Cryopreservation is one of the solutions for fertility restoration in these patients. The cryopreservation of isolated follicles is a more attractive approach in the long term. Many endocrine and paracrine factors can stimulate the granulosa cells of preantral follicles to proliferate. Melatonin acts as direct free radical scavenger and indirect antioxidant. In this study, we investigated the direct effects of melatonin on follicle development and oocyte maturation by exposing in vitro cultured mouse vitrified-warmed ovarian follicles to melatonin. In an experimental study, preantral follicles with diameter of 150-180 micro m were isolated from prepubertal mouse ovaries. Follicles were vitrified and thawed using cryolock method. They were then cultured individually for 7 days in droplets supplemented with 0, 10 and 100 pM melatonin, while ovulation was induced using epidermal growth factor [EGF] and human chorionic gonadotropin [hCG]. The survival rate of follicles and nuclear maturation of ovulated oocytes were determined. At the end of culture, significant increases in follicle survival [p<0.001] and in diameter [p<0.05] were noticed in 10 pM melatonin group compared to control group. In the 100 pM group, survival rate was not affected by melatonin. It was revealed that after induction of ovulation, total number of metaphase II oocytes in treatment groups were not influenced by melatonin [p>0.05]. Culture of mouse vitrified-warmed preantral follicles in a medium supplemented with 10 pM melatonin increased the number of surviving follicles

Vitrification and subsequent in vitro maturation of mouse preantral follicles in presence of growth factors

Cryopreservation of ovarian tissue or follicles has been proposed as an alternative method for fertility preservation. Although successful vitrification of follicles has been reported in several mammalian species, the survival rate is generally low. The aim of this study was to investigate the effects of fibroblast growth factor [FGF] and epidermal growth factor [EGF] on in vitro preantral follicle development after vitrification. In this experimental study, preantral follicles with diameter of 150-180 microm were mechanically isolated from ovaries of 18-21 days old NMRI mice. Follicles were vitrified and warmed, then cultured in alpha-minimal essential medium [alpha-MEM] without growth factor supplementation as control group [group I], while supplemented with 20 ng/ml FGF [group II], 20 ng/ml EGF [group III], and 20 ng/ml FGF +20 ng/ml EGF [group IV]. After 12 days, human chorionic gonadotrophin [hCG]/EGF was added to culture medium, and after 18-20 hours, the presence of cumulus oocyte complexes [COCs] and oocyte maturation were assessed. The chi-square [chi2] test was used to analyze survival and ovulation rates of the follicles. Our results showed that the rate of metaphase II [MII] oocytes in FGF group increased in comparison with control and other treatment groups [p<0.027], but there was no difference between control with EGF and EGF+FGF groups in oocyte maturation rate [p>0.05]. There was a significant decrease in survival rate of follicles in EGF+FGE group in comparison with other groups [p<0.008]. After in vitro ovulation induction, the follicles in EGF group showed a higher ovulation rate [p<0.008] than those cultured in other groups. FGF has beneficial effect on oocyte maturation, and EGF increases COCs number in vitro. Combination of EGF and FGE decreases the number of survived follicles

Melatonin impact on in vitro development of mouse preantral follicles and oocyte maturation

Melatonin acts as an indirect antioxidant and is a powerful direct free radical scavenger and direct responses to melatonin in the gonads are detected. This study aims to investigate the influence of different doses of melatonin on preantral follicle development and oogenesis of in vitro cultured mouse ovarian follicles. Preantral follicles with diameters of 150- 175 micro m were mechanically isolated from NMRI mouse ovaries. Follicles were cultured in droplets of alpha-minimal essential medium [alpha-MEM] supplemented with 5% FBS, 100 mIU/ml rhFSH, 1%ITS, 100 IU/ml penicillin and 100 micro g/ml streptomycin in conjunction with varying doses of melatonin [0, 1, 10, 100 nM and 100, 500 pM] for six days. On day six, in vitro ovulation was induced by the addition of hCG/rEGF to the culture medium and after 16-20 h the maturation state of the oocytes was assessed. There was a significant [P<0.05] decrease in the number of surviving follicles in the groups that received 10, 100 nM and 500 pM melatonin compared to the other groups. After induction of in vitro ovulation, follicles in groups that received 1, 10, and 100 nM melatonin had higher ovulation rates [P<0.05] compared with the other groups. Oocyte maturation capacity was adversely influenced by five concentrations of melatonin and GV arrest was significantly higher compared to the control group [P<0.01]. Our data indicates that a dose of 100 pM melatonin has no toxic effects on follicular development and can be used to reduce oxidative stress in follicle culture systems